The goal of this study was to assess if endogenous 1,25(OH)2D deficiency enhanced, whereas exogenous 1,25(OH)2D3 supplementation alleviated alveolar bone loss and gingival inflammation induced by ligature-induced periodontitis.A model of ligature-induced experimental periodontitis was generated in wild-type (WT) and Cyp27b1-knockout (KO) mice on a rescue diet (RD), and un-ligated genotype-matched littermates as control, or in WT mice on a normal diet (ND) with vehicle treatment or 1,25(OH)2D3 treatment, and un-ligated WT littermates as control. Alveolar bone mass and turnover, T cell infiltration and inflammatory cytokines in gingival tissues were examined.In WT mice, ligature-induced alveolar bone loss occurred by inhibiting alveolar bone formation. This was characterized by reduction of osteoblast numbers, alkaline phosphatase activity and type I collagen synthesis, as well as by augmentation of osteoclastic alveolar bone resorption and gingival inflammation, including increases of osteoclast numbers, inflammatory positive cells and up-regulation of mRNA expression levels of inflammatory cytokines. Alveolar bone destruction and gingival inflammation were more severe in diet-matched Cyp27b1-KO mice than in WT littermates on RD. Supplementation of exogenous 1,25(OH)2D3 alleviated alveolar bone loss and gingival inflammation in ligated WT mice on ND, but those parameters did not reach levels observed in un-ligated WT ones.Endogenous 1,25(OH)2D deficiency enhanced, whereas exogenous 1,25(OH)2D3 supplementation alleviated alveolar bone loss and gingival inflammation induced by ligature-induced periodontitis.