Active vitamin D (1,25 dihydroxyvitamin D or 1,25(OH)2D) exerts its essential action on bone and mineral homeostasis via genomic effects in the intestine, bone and kidney. A critical feature of bone biology is the osteoblast/osteocyte-mediated mineralization of the collagen matrix. While these cells expression the Vitamin D Receptor (VDR) needed to mediate 1,25(OH)2D action, there is conflicting data related to vitamin D’s effect on bone mineralization. We used MLO-A5 cells, a late osteoblast/pre-osteocyte cell model to evaluate the impact of vitamin D signaling on bone mineralization. First, we examined how vitamin D action changed in early osteocytes (100% confluent cultures prior to osteogenic media) and in late osteocytes (6 d in osteogenic media). Cells were treated with 1,25(OH)2D and mRNA was analyzed by qPCR. Cyp24a1 and C3 gene expression showed dose dependent increases to 1,25(OH)2D3 with higher expression after 24 h treatment than 6 h at both cell stages. 1,25(OH)2D3 also increased Enpp1 expression, a key inhibitor of hydroxyapatite formation, in a dose dependent manner but in contrast to Cyp24a1, the induction of Enpp1 was more potent in late osteocytes. Vitamin D-induced expression of Rankl, a promoter of osteoclastogenesis, was dose responsive, but only in early osteocytes prior to mineralization. The impact of 1,25(OH)2D on mineralization after 6 d in osteogenic was directly tested by treating cells for the first 6h or 24h of culture or for the entire 6 d period. There was a dose dependent inhibition of mineralization with continuous exposure to 1,25(OH)2D (-43% at 10 nM, -67% at 100 nM) while early treatment had either no effect (6 h) or a modest impact (-21% at 100 nM 24 h). Because aging reduces bone mass and the predominant cell in bone is osteocytes, we examined whether aging influenced the 1,25(OH)2D-mediated induction of Cyp24a1 mRNA in young (10 wk), mature (20 wk) and old (104 wk) mice (2 ng/g/BW). 1,25(OH)2D increased Cyp24a1 gene expression 10-fold in young mice but there was no induction in old mice (+15%). Overall, our data indicates that 1,25(OH)2D has direct, but stage specific actions on osteocytes to inhibit mineralization. The loss of vitamin D responsiveness with aging may also modify this effect and should be examined further.