Fibroblast growth factor 21 (FGF21) is expressed in several organs, including the liver, adipose tissue, and cardiovascular system, and plays an important role in cross-talk with other organs by binding to specific FGF receptors and their co-receptors. FGF21 represents a potential target for the treatment of obesity, type 2 diabetes mellitus, and non-alcoholic steatohepatitis (NASH). The production of FGF21 in skeletal muscle was recently suggested to be beneficial for metabolic health through its autocrine and paracrine effects. However, the regulatory mechanisms of FGF21 in skeletal muscle remain unclear. In the present study, we showed that berberine regulated FGF21 production in C2C12 myotubes in a dose-dependent manner. We also examined the effects of A-674563, a selective Akt1 inhibitor, on the berberine-mediated regulation of FGF21 expression in C2C12 myotubes. Berberine significantly increased the secretion of FGF21 in C2C12 myotubes, while A-674563 attenuated this effect. Moreover, a pre-treatment with A-674563 effectively suppressed berberine-induced increases in Bmal1 expression in C2C12 myotubes, indicating that the up-regulation of Bmal1 after the berberine treatment was dependent on Akt1. Additionally, berberine-induced increases in FGF21 secretion were significantly attenuated in C2C12 cells transfected with Bmal1 siRNA, indicating the contribution of the core clock transcription factor BMAL1 to Akt-regulated FGF21 in response to berberine. Collectively, these results indicate that berberine regulates the expression of FGF21 through the Akt1 pathway in C2C12 myotubes. Moreover, the core clock gene Bmal1 may participate in the control of the myokine FGF21. Berberine stimulated Akt1-dependent FGF21 expression in C2C12 myotubes. The up-regulation of FGF21 through the modulation of PI3K/AKT1/BMAL1 in response to berberine may be involved in the regulation of cellular function (such as Glut1 expression) by acting in an autocrine and/or paracrine manner in skeletal muscle.