The active form of vitamin D, 1,25(OH)(2)D(3), has a broad range of effects on bone, however, its role in the quality of bone matrix is not well understood. In this study, using an osteoblastic cell (MC3T3-E1) culture system, the effects of 1,25(OH)(2)D(3) on collagen cross-linking and related enzymes, i.e., lysyl hydroxylases (LH1-3) and lysyl oxidases (LOX, LOXL1-4), were examined and compared to controls where cells were treated with cholecalciferol or ethanol. When compared to the controls, gene expressions of LH1, LH2b and LOXL2 were significantly upregulated by 1,25(OH)(2)D(3) up to 72h of culture. In addition, hydroxylysine (Hyl), Hyl aldehyde (Hyl(ald)), Hyl(ald)-derived cross-links and a total number of cross-links of collagen were significantly higher and the cross-link maturation was accelerated in the 1,25(OH)(2)D(3) treated group. These results demonstrate that 1,25(OH)(2)D(3) directly regulates collagen cross-linking in this culture system likely by upregulating gene expression of specific LH and LOX enzymes.