The enzyme β-carotene oxygenase 1 (BCO1) catalyzes the breakdown of provitamin A, including beta-carotene (BC), into retinal, prior to its oxidation into retinoic acid (RA). Allelic variation at the BCO1 locus results in differential expression of its mRNA and affects carotenoid metabolism specifically in chicken Pectoralis major muscle. In this context, the aim of this study was to evaluate the potential myogenic effect of BC and the underlying mechanisms in chicken myoblasts. BCO1 mRNA was detected in myoblasts derived from chicken satellite cells. Treating these myoblasts with BC led to a significant decrease in BrdU incorporation. This anti-proliferative effect was confirmed by a cell cycle study using flow cytometry. BC also significantly increased the differentiation index, suggesting a positive effect on the commitment of avian myoblasts to myogenic differentiation. Addition of DEAB, a specific inhibitor of RALDH activity, significantly reduced BC anti-proliferative and pro-differentiating effects, suggesting that BC exerted its biological effect on chicken myoblasts through activation of the RA pathway. We also observed that in myoblast showing decreased BCO1 expression consecutive to a natural mutation or to a siRNA treatment, the response to BC was inhibited. Nevertheless, BCO1 siRNA transfection increased expression of BCO2 which inhibited cell proliferation in control and BC treated cells.