Obesity is associated with increased risks of diverse diseases; brown adipose tissue (BAT) can increase energy expenditure and protect against obesity by increasing the decomposition of white adipose tissue (WAT) to enhance the non-coupled oxidative phosphorylation of fatty acid in adipocytes and contributes to weight loss. However, BAT is abundant in only small rodents and newborn humans, but not in adults. PRDM16 is a key factor that induces the differentiation of skeletal muscle precursors to brown adipocytes and simultaneously inhibits myogenic differentiation. In the present study, we set insulin-induced skeletal muscle satellite cells (SMSCs) adipogenic differentiation model, as confirmed by the contents of adipogenic markers PRDM16, UCP1 and PGC1α and myogenic markers MyoD1 and MyoG. We selected miR-499 as candidate miRNA, which might regulate PRDM16 to affect SMSCs adipogenic differentiation. Possibly through directly binding to PRDM16 3'-UTR, miR-499 negatively regulated PRDM16 expression and hindered SMSCs adipogenic differentiation by reducing adipogenic markers PRDM16, UCP1 and PGC1α and increasing myogenic markers MyoD1 and MyoG. PRDM16 overexpression could partially reverse the effect of miR-499 on the above markers and SMSCs adipogenic differentiation. Taken together, miR-499/PRDM16 axis can affect the balance between SMSC myogenic and adipogenic differentiation, targeting miR-499 to rescue PRDM16 expression, thus promoting SMSCs adipogenic differentiation may be a promising strategy for obesity treatment.