The CC genotype of the vitamin D receptor (VDR) polymorphism TaqI rs731236 has previously been associated with a higher risk of developing myopathy compared to TT carriers. However, the mechanistic role of this polymorphism in skeletal muscle is not well defined. The effects of vitamin D on patients genotyped for the VDR polymorphism TaqI rs731236, comparing CC and TT carriers were evaluated. Primary human myoblasts isolated from 4 CC carriers were compared with myoblasts isolated from four TT carriers and treated with vitamin D in vitro. A dose-dependent inhibitory effect on myoblast proliferation and differentiation was observed concurrent with modifications of key myogenic regulatory factors. RNA sequencing revealed a vitamin D dose–response gene signature enriched with a higher number of VDR-responsive elements (VDREs) per gene. Interestingly, the greater the expression of muscle differentiation markers in myoblasts, the more pronounced was the vitamin D-mediated response to suppress genes associated with myogenic fusion and myotube formation. This novel finding provides a mechanistic explanation to the inconsistency regarding previous reports of the role of vitamin D in myoblast differentiation. No effects in myoblast proliferation, differentiation or gene expression were related to CC vs TT carriers. Our findings suggest that the VDR polymorphism TaqI rs731236 comparing CC vs TT carriers did not influence the effects of vitamin D on primary human myoblasts and that vitamin D inhibits myoblast proliferation and differentiation through key regulators of cell cycle progression. Future studies need to employ strategies to identify the primary responses of vitamin D that drive the cellular response towards quiescence.