diff --git a/docs/source/conf.py b/docs/source/conf.py
index c20869e12..d385630d2 100644
--- a/docs/source/conf.py
+++ b/docs/source/conf.py
@@ -87,7 +87,6 @@
 
 nitpicky = True
 nitpick_ignore = [('py:class', 'Intracomm'),
-                  ('py:class', 'h5py.RegionReference'),
                   ('py:class', 'h5py._hl.dataset.Dataset'),
                   ('py:class', 'function'),
                   ('py:class', 'unittest.case.TestCase'),
diff --git a/src/hdmf/backends/hdf5/h5tools.py b/src/hdmf/backends/hdf5/h5tools.py
index bf8f86bb9..a6a4e4a25 100644
--- a/src/hdmf/backends/hdf5/h5tools.py
+++ b/src/hdmf/backends/hdf5/h5tools.py
@@ -1492,7 +1492,7 @@ def __is_ref(self, dtype):
         if isinstance(dtype, dict):  # may be dict from reading a compound dataset
             return self.__is_ref(dtype['dtype'])
         if isinstance(dtype, str):
-            return dtype == DatasetBuilder.OBJECT_REF_TYPE or dtype == DatasetBuilder.REGION_REF_TYPE
+            return dtype == DatasetBuilder.OBJECT_REF_TYPE
         return False
 
     def __queue_ref(self, func):
diff --git a/src/hdmf/build/__init__.py b/src/hdmf/build/__init__.py
index ea5d21152..87e0ac57e 100644
--- a/src/hdmf/build/__init__.py
+++ b/src/hdmf/build/__init__.py
@@ -1,4 +1,4 @@
-from .builders import Builder, DatasetBuilder, GroupBuilder, LinkBuilder, ReferenceBuilder, RegionBuilder
+from .builders import Builder, DatasetBuilder, GroupBuilder, LinkBuilder, ReferenceBuilder
 from .classgenerator import CustomClassGenerator, MCIClassGenerator
 from .errors import (BuildError, OrphanContainerBuildError, ReferenceTargetNotBuiltError, ContainerConfigurationError,
                      ConstructError)
diff --git a/src/hdmf/build/objectmapper.py b/src/hdmf/build/objectmapper.py
index c2ef44b5f..176de322c 100644
--- a/src/hdmf/build/objectmapper.py
+++ b/src/hdmf/build/objectmapper.py
@@ -6,7 +6,7 @@
 
 import numpy as np
 
-from .builders import DatasetBuilder, GroupBuilder, LinkBuilder, Builder, ReferenceBuilder, RegionBuilder, BaseBuilder
+from .builders import DatasetBuilder, GroupBuilder, LinkBuilder, Builder, ReferenceBuilder, BaseBuilder
 from .errors import (BuildError, OrphanContainerBuildError, ReferenceTargetNotBuiltError, ContainerConfigurationError,
                      ConstructError)
 from .manager import Proxy, BuildManager
@@ -1214,8 +1214,6 @@ def __get_subspec_values(self, builder, spec, manager):
                 continue
             if isinstance(attr_val, (GroupBuilder, DatasetBuilder)):
                 ret[attr_spec] = manager.construct(attr_val)
-            elif isinstance(attr_val, RegionBuilder):  # pragma: no cover
-                raise ValueError("RegionReferences as attributes is not yet supported")
             elif isinstance(attr_val, ReferenceBuilder):
                 ret[attr_spec] = manager.construct(attr_val.builder)
             else:
diff --git a/src/hdmf/validate/validator.py b/src/hdmf/validate/validator.py
index daa5adac4..d7ec78eaa 100644
--- a/src/hdmf/validate/validator.py
+++ b/src/hdmf/validate/validator.py
@@ -8,7 +8,7 @@
 
 from .errors import Error, DtypeError, MissingError, MissingDataType, ShapeError, IllegalLinkError, IncorrectDataType
 from .errors import ExpectedArrayError, IncorrectQuantityError
-from ..build import GroupBuilder, DatasetBuilder, LinkBuilder, ReferenceBuilder, RegionBuilder
+from ..build import GroupBuilder, DatasetBuilder, LinkBuilder, ReferenceBuilder
 from ..build.builders import BaseBuilder
 from ..spec import Spec, AttributeSpec, GroupSpec, DatasetSpec, RefSpec, LinkSpec
 from ..spec import SpecNamespace
@@ -124,9 +124,6 @@ def get_type(data, builder_dtype=None):
     # Bytes data
     elif isinstance(data, bytes):
         return 'ascii', get_string_format(data)
-    # RegionBuilder data
-    elif isinstance(data, RegionBuilder):
-        return 'region', None
     # ReferenceBuilder data
     elif isinstance(data, ReferenceBuilder):
         return 'object', None
diff --git a/tests/unit/build_tests/test_builder.py b/tests/unit/build_tests/test_builder.py
index a35dc64ac..62ebd0675 100644
--- a/tests/unit/build_tests/test_builder.py
+++ b/tests/unit/build_tests/test_builder.py
@@ -1,4 +1,4 @@
-from hdmf.build import GroupBuilder, DatasetBuilder, LinkBuilder, ReferenceBuilder, RegionBuilder
+from hdmf.build import GroupBuilder, DatasetBuilder, LinkBuilder, ReferenceBuilder
 from hdmf.testing import TestCase
 
 
@@ -392,12 +392,3 @@ def test_constructor(self):
         db = DatasetBuilder('db1', [1, 2, 3])
         rb = ReferenceBuilder(db)
         self.assertIs(rb.builder, db)
-
-
-class TestRegionBuilder(TestCase):
-
-    def test_constructor(self):
-        db = DatasetBuilder('db1', [1, 2, 3])
-        rb = RegionBuilder(slice(1, 3), db)
-        self.assertEqual(rb.region, slice(1, 3))
-        self.assertIs(rb.builder, db)
diff --git a/tests/unit/spec_tests/test_ref_spec.py b/tests/unit/spec_tests/test_ref_spec.py
index bb1c0efb8..3277673d1 100644
--- a/tests/unit/spec_tests/test_ref_spec.py
+++ b/tests/unit/spec_tests/test_ref_spec.py
@@ -15,9 +15,3 @@ def test_constructor(self):
     def test_wrong_reference_type(self):
         with self.assertRaises(ValueError):
             RefSpec('TimeSeries', 'unknownreftype')
-
-    def test_isregion(self):
-        spec = RefSpec('TimeSeries', 'object')
-        self.assertFalse(spec.is_region())
-        spec = RefSpec('Data', 'region')
-        self.assertTrue(spec.is_region())