BACKGROUND: Although the role of vitamin D receptor (VDR) in muscle mass and strength is well established, the effects of vitamin D (VD) on muscle remain controversial due to various factors. Herein, the influence of sex on the effects of VD on muscle function and the underlying reasons was explored. METHODS: Male and female Sod1 gene knockout (SKO) mice, serving as a model for skeletal muscle atrophy, were treated with the VD active analogue calcipotriol, and RNA sequencing was employed to investigate this potential signalling pathway. The National Health and Nutrition Examination Survey (NHANES) database was utilized to explore whether testosterone affects the correlation between VD and grip strength in human participants. Experiments involving C2C12 cells and castrated male mice subjected to immobilization were conducted to demonstrate the enhancing effects of testosterone on VD function. RESULTS: In male SKO mice, Vdr expression in the gastrocnemius muscle was positively correlated with grip strength (R CONCLUSIONS: This study demonstrates the existence of sex heterogeneity in the effects of VD on muscle and that testosterone enhances the strength and molecular responses to VD. These findings underscore the importance of considering testosterone levels when utilizing VD to enhance muscle strength.