Background: Vitamin K derivatives have an important role in bone formation and blood clotting. Vitamin K₂ [menaquinone-4 (MK-4)] is used for the treatment and prevention of osteoporosis. Recently, vitamin K was found to be effective for the prevention of osteoarthritis and may play a role in cartilage formation; however, the function of UBIAD1 (MK-4 biosynthesis enzyme) and MK-4 in cartilage is unclear. In this study, we examined the function of UBIAD1 and MK-4 in chondrogenesis and differentiation using chondrogenically differentiated cells. Methods: Mouse chondrocyte progenitor ATDC5 cells were used for siRNA knockdown of UBIAD1 to determine its effects on cell proliferation and chondrogenic differentiation. Proliferation and differentiation were assessed using the WST-8 assay and Alcian blue staining, respectively. The effects of MK-4 treatment and transfection of a human UBIAD1 expression plasmid on UBIAD1 knockdown cells were also examined. RESULTS: UBIAD1 knockdown significantly decreased the proliferation and chondrogenic differentiation of ATDC5 cells. MK-4 treatment suppressed ATDC5 cell proliferation and chondrogenic differentiation. It also affected UBIAD1 knockdown ATDC5 cells during chondrogenic differentiation. However, overexpression of human UBIAD1 promoted ATDC5 cell proliferation and chondrogenic differentiation and exhibited a similar effect on UBIAD1 knockdown ATDC5 cells. These results suggest that MK-4 has an inhibitory effect on the proliferation and differentiation of UBIAD1, which exhibits a promoting effect. These results suggest that UBIAD1 and MK-4 have a role in regulating chondrocyte proliferation and differentiation and are important regulators of chondrogenic differentiation.