diff --git a/localstack/config.py b/localstack/config.py
index 7f254706fe071..51bcf1f7f8fe6 100644
--- a/localstack/config.py
+++ b/localstack/config.py
@@ -430,9 +430,6 @@ def in_docker():
 # Whether to report internal failures as 500 or 501 errors.
 FAIL_FAST = is_env_true("FAIL_FAST")
 
-# whether to use the legacy single-region mode, defined via DEFAULT_REGION (DEPRECATED!)
-USE_SINGLE_REGION = is_env_true("USE_SINGLE_REGION")
-
 # whether to run in TF compatibility mode for TF integration tests
 # (e.g., returning verbatim ports for ELB resources, rather than edge port 4566, etc.)
 TF_COMPAT_MODE = is_env_true("TF_COMPAT_MODE")
@@ -1283,7 +1280,7 @@ def use_custom_dns():
     "SYNCHRONOUS_SNS_EVENTS",
     "TEST_AWS_ACCOUNT_ID",
     "TF_COMPAT_MODE",
-    "USE_SINGLE_REGION",
+    "USE_SINGLE_REGION",  # Not functional; deprecated in 0.12.7, removed in 3.0.0
     "USE_SSL",
     "WAIT_FOR_DEBUGGER",
     "WINDOWS_DOCKER_MOUNT_PREFIX",
diff --git a/localstack/deprecations.py b/localstack/deprecations.py
index d54c16f2b056a..6850b32127c46 100644
--- a/localstack/deprecations.py
+++ b/localstack/deprecations.py
@@ -47,11 +47,11 @@ def is_affected(self) -> bool:
         "0.12.8",
         "PORT_WEB_UI has been removed, and is not available anymore. Please remove this environment variable.",
     ),
-    # Since 0.12.7 - Full multi-region support
+    # Deprecated in 0.12.7, removed in 3.0.0
     EnvVarDeprecation(
         "USE_SINGLE_REGION",
         "0.12.7",
-        "LocalStack now has full multi-region support. Please remove this environment variable.",
+        "LocalStack now has full multi-region support. This option has no effect. Please remove it from your configuration.",  # noqa
     ),
     EnvVarDeprecation(
         "DEFAULT_REGION",
diff --git a/localstack/runtime/analytics.py b/localstack/runtime/analytics.py
index bc3ebb2a074b5..7c05c2060a64b 100644
--- a/localstack/runtime/analytics.py
+++ b/localstack/runtime/analytics.py
@@ -37,7 +37,7 @@
     "REQUIRE_PRO",
     "SKIP_INFRA_DOWNLOADS",
     "SQS_ENDPOINT_STRATEGY",
-    "USE_SINGLE_REGION",
+    "USE_SINGLE_REGION",  # Not functional; deprecated in 0.12.7, removed in 3.0.0
     "USE_SSL",
 ]
 
diff --git a/localstack/services/edge.py b/localstack/services/edge.py
index efe4ffa9a4d85..4f77bb6034095 100644
--- a/localstack/services/edge.py
+++ b/localstack/services/edge.py
@@ -41,7 +41,6 @@
 from localstack.utils.aws.aws_stack import (
     extract_access_key_id_from_auth_header,
     is_internal_call_context,
-    set_default_region_in_headers,
 )
 from localstack.utils.collections import split_list_by
 from localstack.utils.functions import empty_context_manager
@@ -125,8 +124,6 @@ def forward_request(self, method, path, data, headers):
         if api:
             port = get_service_port_for_account(api, headers)
 
-        set_default_region_in_headers(headers)
-
         should_log_trace = is_trace_logging_enabled(headers)
         if api and should_log_trace:
             # print request trace for debugging, if enabled
diff --git a/localstack/utils/aws/aws_stack.py b/localstack/utils/aws/aws_stack.py
index c80b04f7ef6ec..fc9420e4f82b8 100644
--- a/localstack/utils/aws/aws_stack.py
+++ b/localstack/utils/aws/aws_stack.py
@@ -200,24 +200,6 @@ def get_s3_hostname():
     return LOCALHOST
 
 
-def set_default_region_in_headers(headers, service=None, region=None):
-    # this should now be a no-op, as we support arbitrary regions and don't use a "default" region
-    # TODO: remove this function once the legacy USE_SINGLE_REGION config is removed
-    if not config.USE_SINGLE_REGION:
-        return
-
-    auth_header = headers.get("Authorization")
-    region = region or get_region()
-    if not auth_header:
-        if service:
-            headers["Authorization"] = mock_aws_request_headers(service, region_name=region)[
-                "Authorization"
-            ]
-        return
-    replaced = re.sub(r"(.*Credential=[^/]+/[^/]+/)([^/])+/", r"\1%s/" % region, auth_header)
-    headers["Authorization"] = replaced
-
-
 def fix_account_id_in_arns(response, colon_delimiter=":", existing=None, replace=None):
     """Fix the account ID in the ARNs returned in the given Flask response or string"""
     existing = existing or ["123456789", "1234567890", "123456789012", get_aws_account_id()]
diff --git a/localstack/utils/aws/request_context.py b/localstack/utils/aws/request_context.py
index 817f680cecdf6..e4485ef21288d 100644
--- a/localstack/utils/aws/request_context.py
+++ b/localstack/utils/aws/request_context.py
@@ -107,9 +107,6 @@ def __exit__(self, type, value, traceback):
 def get_region_from_request_context():
     """look up region from request context"""
 
-    if config.USE_SINGLE_REGION:
-        return
-
     request_context = get_request_context()
     if not request_context:
         return
@@ -197,9 +194,6 @@ def convert_to_flask_response_call(fn, *args, **kwargs):
             # TODO: publish analytics event ...
             return requests_to_flask_response(response)
 
-    if config.USE_SINGLE_REGION:
-        return
-
     # make sure that we inherit THREAD_LOCAL request contexts to spawned sub-threads
     @patch(FuncThread.__init__)
     def thread_init(fn, self, *args, **kwargs):