The vitamin K family members, phylloquinones (vitamin K1) and the menaquinones (vitamin K2), are currently under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologues, phytonadione (K1) and menatetrenone (K2), and those of the synthetic vitamin K, menadione (K3), on human primary osteoblasts. All homologues promoted in vitro mineralization by these cells. K1-induced mineralization was highly sensitive to warfarin, whereas that induced by K2 and K3 was less sensitive, implying that both -carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor (SXR), are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of K2 or K3, in a 3-dimensional (3D) type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K treated cells revealed vitamin K dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the transition of human osteoblasts into osteocytes. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K treatment reduced RANKL expression relative to OPG by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the transition of osteoblasts into osteocytes, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms, by which vitamin K optimizes bone formation and integrity in vivo, and may help explain the net positive effect of vitamin K on bone formation.