Vitamin K2 is a critical nutrient required for blood coagulation. It also plays a key role in bone homeostasis and is a clinically effective therapeutic agent for osteoporosis. We previously demonstrated that vitamin K2 is a transcriptional regulator of bone marker genes in osteoblastic cells and that it may potentiate bone formation by activating the steroid and xenobiotic receptor, SXR. To explore the SXR-mediated vitamin K2 signaling network in bone homeostasis, we identified genes up-regulated by both vitamin K2 and the prototypical SXR ligand, rifampicin, in osteoblastic cells using oligonucleotide microarray analysis and quantitative reverse transcription-PCR. Fourteen genes were up-regulated by both ligands. Among these, tsukushi, matrilin-2, and CD14 antigen were shown to be primary SXR target genes. Moreover, collagen accumulation in osteoblastic MG63 cells was enhanced by vitamin K2 treatment. Gain- and loss-of-function analyses showed that the small leucine-rich proteoglycan, tsukushi, contributes to vitamin K2-mediated enhancement of collagen accumulation. Our results suggest a new function for vitamin K2 in bone formation as a transcriptional regulator of extracellular matrix-related genes, that are involved in the collagen assembly.