Retinoic acid receptor-related orphan receptor (ROR) α has been implicated in various physiological functions, including the immune system, inflammation, and circadian rhythms. In the present study, the synthetic RORα/γ agonist SR1078 stimulated the production and gene expression of fibroblast growth factor 21 (FGF21) in C2C12 myotubes. FGF21, a member of the FGF family, plays important roles in the regulation of peripheral glucose tolerance and lipid metabolism and improves metabolic health. The mRNA expression and secretion of FGF21 was significantly weaker in Rora-silenced cells than in cells transfected with non-targeting control siRNA. SR1078 significantly up-regulated C/EBP homologous protein (CHOP), an established marker of ER stress, in a dose-dependent manner in C2C12 myotubes, while CHOP expression was decreased in Rora-silenced C2C12 cells, suggesting that RORα is involved in the regulation of FGF21 expression and stimulates ER stress in C2C12 myotubes. The naturally occurring compound baicalein up-regulated FGF21 expression and secretion in C2C12 myotubes. Additionally, the up-regulation of CHOP mRNA and protein expression was observed in C2C12 myotubes after the baicalein treatment. Furthermore, the knockdown of RORα prevented the augmentation of FGF21 and up-regulation of CHOP in response to baicalein in C2C12 cells. Collectively, these results suggest that baicalein stimulates the ER stress response and FGF21 expression through an RORα-dependent mechanism in C2C12 myotubes, and indicate the potential of baicalein as an effective anti-obesity therapy via its ability to enhance FGF21 production.