This study aimed at the preparation of a sustained-release 25-hydroxyvitamin D(3) (25OHD) treatment for diabetic periodontitis, a known complication of diabetes. 25OHD-loaded polylactic acid (PLA) microspheres were prepared using oil-in-water emulsion-solvent evaporation method. The prepared microspheres exhibited intact surfaces, with average sizes ranging from 42.3 to 119.4 μm. The encapsulation efficiency ranged from 79.2% (w/w) to 88.5% (w/w), and the drug content was between 15.8% (w/w) and 17.8% (w/w). Drug release from the produced microspheres followed a near-to-zero-order release pattern and lasted over 10 weeks. In an in vitro model of diabetic periodontitis, the abnormal morphological changes and the decrease in the cell viability of bone marrow stromal cells could be effectively attenuated after the 25OHD-loaded microsphere application. Additionally, in a rat model of diabetic periodontitis, alveolar bone loss was inhibited and osteoid formation in the periodontium was promoted upon 25OHD-loaded microsphere treatment. In conclusion, 25OHD-loaded PLA microspheres may provide an effective approach for the treatment of this disease.