Type 2 diabetic mellitus is manifested by metabolic impairments with high prevalence worldwide, of which periodontitis represents a typical oral complication (also called diabetic periodontitis). Oral epithelia bear the brunt of periodontal damage from microscopic intruders; thus the defense function of epithelial cells is of vital significance. We have previously proved that 25-hydroxyvitamin D₃ (25-OHD₃) altered the expression of cathelicidin antimicrobial peptide in oral epithelial cells in vitro. Herein, we discovered that 25-OHD₃ intraperitoneal injection attenuated periodontal inflammation by promoting cathelicidin production in gingival epithelia and reducing fasting glucose of diabetic mice. Dotblotting of serum showed cathelicidin secretion was consistent with 25-OHD₃ treatment. Immunochemistry exhibited enhanced expression of cathelicidin and vitamin D receptors along with reduced expression of TLR4 in diabetic mice. Stereomicroscope showed less alveolar bone loss when injected with 25-OHD₃.These results showed 25-OHD₃ can promote cathelicidin and ameliorate the severity of diabetic periodontitis. Our study complemented the mechanism of cathelicidin and extended knowledge of 25-OHD₃'s role in diabetic periodontitis.