diff --git a/moto/dynamodb2/exceptions.py b/moto/dynamodb2/exceptions.py
index 5e7d63b79..31514923f 100644
--- a/moto/dynamodb2/exceptions.py
+++ b/moto/dynamodb2/exceptions.py
@@ -198,6 +198,13 @@ class TransactionCanceledException(ValueError):
         super().__init__(msg)
 
 
+class MultipleTransactionsException(MockValidationException):
+    msg = "Transaction request cannot include multiple operations on one item"
+
+    def __init__(self):
+        super().__init__(self.msg)
+
+
 class EmptyKeyAttributeException(MockValidationException):
     empty_str_msg = "One or more parameter values were invalid: An AttributeValue may not contain an empty string"
     # AWS has a different message for empty index keys
diff --git a/moto/dynamodb2/models/__init__.py b/moto/dynamodb2/models/__init__.py
index adb51a1c7..6c03482c7 100644
--- a/moto/dynamodb2/models/__init__.py
+++ b/moto/dynamodb2/models/__init__.py
@@ -23,6 +23,7 @@ from moto.dynamodb2.exceptions import (
     TransactionCanceledException,
     EmptyKeyAttributeException,
     InvalidAttributeTypeError,
+    MultipleTransactionsException,
 )
 from moto.dynamodb2.models.utilities import bytesize
 from moto.dynamodb2.models.dynamo_type import DynamoType
@@ -1566,6 +1567,14 @@ class DynamoDBBackend(BaseBackend):
     def transact_write_items(self, transact_items):
         # Create a backup in case any of the transactions fail
         original_table_state = copy.deepcopy(self.tables)
+        target_items = set()
+
+        def check_unicity(table_name, key):
+            item = (str(table_name), str(key))
+            if item in target_items:
+                raise MultipleTransactionsException()
+            target_items.add(item)
+
         errors = []
         for item in transact_items:
             try:
@@ -1573,6 +1582,7 @@ class DynamoDBBackend(BaseBackend):
                     item = item["ConditionCheck"]
                     key = item["Key"]
                     table_name = item["TableName"]
+                    check_unicity(table_name, key)
                     condition_expression = item.get("ConditionExpression", None)
                     expression_attribute_names = item.get(
                         "ExpressionAttributeNames", None
@@ -1611,6 +1621,7 @@ class DynamoDBBackend(BaseBackend):
                     item = item["Delete"]
                     key = item["Key"]
                     table_name = item["TableName"]
+                    check_unicity(table_name, key)
                     condition_expression = item.get("ConditionExpression", None)
                     expression_attribute_names = item.get(
                         "ExpressionAttributeNames", None
@@ -1629,6 +1640,7 @@ class DynamoDBBackend(BaseBackend):
                     item = item["Update"]
                     key = item["Key"]
                     table_name = item["TableName"]
+                    check_unicity(table_name, key)
                     update_expression = item["UpdateExpression"]
                     condition_expression = item.get("ConditionExpression", None)
                     expression_attribute_names = item.get(
@@ -1648,6 +1660,10 @@ class DynamoDBBackend(BaseBackend):
                 else:
                     raise ValueError
                 errors.append(None)
+            except MultipleTransactionsException:
+                # Rollback to the original state, and reraise the error
+                self.tables = original_table_state
+                raise MultipleTransactionsException()
             except Exception as e:  # noqa: E722 Do not use bare except
                 errors.append(type(e).__name__)
         if any(errors):
diff --git a/moto/dynamodb2/responses.py b/moto/dynamodb2/responses.py
index e1d669c58..56115ba4b 100644
--- a/moto/dynamodb2/responses.py
+++ b/moto/dynamodb2/responses.py
@@ -1127,6 +1127,9 @@ class DynamoHandler(BaseResponse):
         except TransactionCanceledException as e:
             er = "com.amazonaws.dynamodb.v20111205#TransactionCanceledException"
             return self.error(er, str(e))
+        except MockValidationException as mve:
+            er = "com.amazonaws.dynamodb.v20111205#ValidationException"
+            return self.error(er, mve.exception_msg)
         response = {"ConsumedCapacity": [], "ItemCollectionMetrics": {}}
         return dynamo_json_dump(response)
 
diff --git a/tests/test_dynamodb2/exceptions/test_dynamodb_exceptions.py b/tests/test_dynamodb2/exceptions/test_dynamodb_exceptions.py
index 61b5e0354..7f0b623b9 100644
--- a/tests/test_dynamodb2/exceptions/test_dynamodb_exceptions.py
+++ b/tests/test_dynamodb2/exceptions/test_dynamodb_exceptions.py
@@ -1,12 +1,10 @@
 import boto3
 import pytest
 import sure  # noqa # pylint: disable=unused-import
-
-from botocore.exceptions import ClientError
 from boto3.dynamodb.conditions import Key
+from botocore.exceptions import ClientError
 from moto import mock_dynamodb2
 
-
 table_schema = {
     "KeySchema": [{"AttributeName": "partitionKey", "KeyType": "HASH"}],
     "GlobalSecondaryIndexes": [
@@ -466,3 +464,41 @@ def test_creating_table_with_0_global_indexes():
     err["Message"].should.equal(
         "One or more parameter values were invalid: List of GlobalSecondaryIndexes is empty"
     )
+
+
+@mock_dynamodb2
+def test_multiple_transactions_on_same_item():
+    table_schema = {
+        "KeySchema": [{"AttributeName": "id", "KeyType": "HASH"}],
+        "AttributeDefinitions": [{"AttributeName": "id", "AttributeType": "S"},],
+    }
+    dynamodb = boto3.client("dynamodb", region_name="us-east-1")
+    dynamodb.create_table(
+        TableName="test-table", BillingMode="PAY_PER_REQUEST", **table_schema
+    )
+    # Insert an item
+    dynamodb.put_item(TableName="test-table", Item={"id": {"S": "foo"}})
+
+    def update_email_transact(email):
+        return {
+            "Update": {
+                "Key": {"id": {"S": "foo"}},
+                "TableName": "test-table",
+                "UpdateExpression": "SET #e = :v",
+                "ExpressionAttributeNames": {"#e": "email_address"},
+                "ExpressionAttributeValues": {":v": {"S": email}},
+            }
+        }
+
+    with pytest.raises(ClientError) as exc:
+        dynamodb.transact_write_items(
+            TransactItems=[
+                update_email_transact("test1@moto.com"),
+                update_email_transact("test2@moto.com"),
+            ]
+        )
+    err = exc.value.response["Error"]
+    err["Code"].should.equal("ValidationException")
+    err["Message"].should.equal(
+        "Transaction request cannot include multiple operations on one item"
+    )