Abstract: Objective: To investigate the bone health improvement effects and mechanism of vitamin K₂ alone and in combination with calcium on osteoporotic zebrafish model. Methods: Healthy zebrafish with transgenic Tg line were used as experimental animals. The model of osteoporosis zebrafish was induced by prednisone. The fluorescence area and density of zebrafish osteoblasts were used as indicators to evaluate the bone development of zebrafish which were observed and photographed under microscope. Furthermore, the expression changes of related genes were detected by Real-time qPCR. Results: Vitamin K₂ alone and in combination with D₃ and calcium chloride could significantly improve the reduction of fluorescence area and fluorescence density of zebrafish osteoblasts induced by prednisolone in the experimental safe dose range of 5~20 μg/mL (P<0.05). Vitamin K₂ played anti-osteoporosis activity by promoting the expression of alp, bmp2a and bmp4 genes related to osteoblast, inhibiting the expression of opg, rank1, trap and ctsk genes related to osteoclast, and inhibiting the expression of vdrb, sparc and colla2 genes related to bone extracellular matrix. Conclusion: Vitamin K₂ could improve the bone health of zebrafish by promoting the expression of genes related to osteoblast formation and inhibit the expression of genes related to osteoclasts and bone calcification in extracellular matrix. The combined administration of vitamin K₂ with D₃ and calcium chloride could enhance the ability to improve bone health.