Purpose: Age-related loss of skeletal muscle mass (sarcopenia) and the associated loss of muscle function (dynapenia) is a major concern for the healthcare system. Indeed, as the sheer number of elderly individuals rises due to increases in longevity, as a result of improved healthcare and the post-war “baby-boom”, age related frailty is an increasingly onerous problem of complex etiology. Epidemiological studies have linked Vitamin D deficiency to, age-related declines in muscle function and regenerative capacity in addition reduced muscle protein expression of the Vitamin D receptor (VDR) (1). Moreover, bona fide confirmation of VDR expression in muscle cells has only recently been substantiated. In recent studies, muscle VDR expression has been positively linked to tissue regeneration (2) and also to be under the control of expression by exogenous vitamin D3. In this study, we hypothesized that the VDR has a mechanistic role at multiple levels of myogenic regulation. Results: In order to study the autonomous role of VDR we generated C2C12 skeletal muscle cells harboring sustained shRNA lentiviral-mediated knockdown of the VDR (VDR-KD). Knockdown was confirmed such that VDR-KD cells exhibited <85% of VDR expression vs. scrambled sequence shRNA (SCR) transfected cells. VDR-KD cells proliferated at a slower rate than SCR cells (-27±6%, P<0.01) displaying a reduction in DNA synthesis (assessed via BRDU incorporation) (-31±7%, P<0.05). Furthermore, altered cell-cycle activities, as assayed by flow cytometry, were also evident in VDR-KD cells, with a greater proportion of the cell population being G0/G1 phase (+12±6%, P<0.05). Following induction of differentiation total alkaline soluble protein was measured at multiple days, with VDR-KD populations shown to be consistently (~20%±3%) less than SCR controls. During differentiation SCR populations exhibited no significant increases in total DNA content, whereas VDR-KD content continued to increase (SCR +26±6% N.S. total DNA vs. VDR-KD +325±26% P<0.05). Myosin protein expression throughout differentiation was markedly reduced in VDR-KD vs. SCR cells (-89±2%, P<0.001). VDR-KD cells impaired differentiation characteristics, producing fewer myotubes (SCR 24±1/mm2 vs. VDR 18±1, P<0.01, N=6) with a greater diameter compared to SCR controls (+10±2% P<0.05, N=6). In addition a greater myonuclei number was observed in VDR-KD myotubes (SCR 10±0.3 nuclei/myotube vs. VDR 25±1, P<0.05). Conclusion: The VDR plays a role in the myogenic regulation of myoblast proliferation and differentiation suggesting a fundamental role of the VDR in the control of myogenesis, and perhaps mass and function. These findings imply an autonomous role of the VDR and may explain potentially formative links between vitamin D, the VDR, and impairments of muscle mass/ function and metabolism in ageing and any associated vitamin D deficient conditions.