Vitamin D deficiency is associated with a decline in muscle function and an increasing risk of muscle injury in athletes and the elderly. Nevertheless, how vitamin D3 and vitamin D receptor (VDR) regulate skeletal muscle cells under pro-fibrotic factor stimulation that could be pronounced during repetitive muscle damage have not been elucidated. Therefore, this study aimed to investigate the regulatory role of cholecalciferol (D3), calcidiol (25D3), calcitriol (1,25D3), and the effect of Vdr gene suppression under TGF-β1 stimulation in C2C12 mouse skeletal muscle cells. All forms of vitamin D3 exerted anti-fibrotic effects under TGF-β1 stimulation by suppression of COL1A1; however, D3 preserves this effect without a negative impact on myogenesis. Moreover, LC-MS/MS-based proteomics analysis revealed that myoblast fusion protein and mitochondrial regulation were altered following Vdr knockdown. These changes were associated with exacerbation of α-SMA expression in TGF-β1-treated cells and suggested VDR modulates fibrogenesis in skeletal muscle cells regardless of ligand binding. Under TGF-β1 stimulation, anti-fibrotic effects of 1,25D3 but not D3 were diminished after Vdr knockdown, supporting D3 action is not dependent on VDR activation. Collectively, understanding anti-fibrotic effects of vitamin D3 is beneficial for providing a strategy of vitamin D supplementation to counteract fibrosis development after muscle injury.