To investigate the inhibitory effects of 25-hydroxyvitamin D3 (25(OH)D3)-loaded polylactic acid (PLA) microspheres on inflammatory response in diabetic periodontitis. 25(OH)D3-loaded PLA microspheres were produced using emulsion-solvent evaporation method. Rat bone marrow stromal cells (BMSCs) cultured with Aggregatibacter actinomycetemcomitans in high glucose medium were chosen to mimic diabetic periodontitis. After cultivation with 25(OH)D3-loaded PLA microspheres, inflammation-related proteins in BMSCs were detected using Western blot analysis. Periodontitis was induced using silk ligatures in diabetic rats, and 25(OH)D3-loaded PLA microspheres were placed into the periodontal pockets. Periodontal tissues were examined using hematoxylin and eosin staining and western blot analysis. Drug release from the 25(OH)D3-loaded microspheres was relatively steady during 70 days. In a diabetic periodontitis-like environment, 25(OH)D3-loaded microspheres upregulated vitamin D receptor expression, and downregulated nuclear factor-κB expression and signal transducer and activator of transcription 3 phosphorylation in the BMSCs. These 25(OH)D3 microspheres also attenuated periodontal inflammatory infiltrate and bone loss in diabetic rats with periodontitis. 25(OH)D3-loaded microspheres could ameliorate diabetic periodontitis by inhibiting inflammatory response, and may provide a potential therapy for patients with this disease.