Purpose: Fibronectin type Ⅲ domaincontaining protein 5 (FNDC5), also known as irisin, is a myokine secreted from muscle in response to exercise and improves obesity and glucose homeostasis.However, the molecular mechanisms that regulate FNDC5 expression and the functional significance of FNDC5 in skeletal muscle remain unknown.In this study, we explored the possible pathways that induce FNDC5 expression and delineated its metabolic effects on skeletal muscle.Methods: C2C12 myotubes were treated with various concentrations of SpcAMP,forskolin, and ionomycin respectively for various durations.FNDC5 and related metabolic genes expressions were measured by quantitative realtime polymerase chain reaction (qRTPCR).Cyclic AMP responsive elementbinding protein (CREB) phosphorylation was measured by Western blot.Oxidative phosphorylation was quantified by oxygen consumption rate (OCR) measurement using XF96 analyzer (Seahorse Bioscience).The statistical significance was calculated by oneway analysis of variance (ANOVA).Data were considered significant when P ＜ 0.05.Results: We found that cAMP and forskolin dose and time dependently increased FNDC5 expression in C2C12myotubes.A synergistic effect of forskolin and ionomycin on FNDC5 expression was also found.CREB phosphorylation was elevated in myotubes simultaneously upon these treatments.C2C12 myotubes over expressing CREB displays increased FNDC5 expression as well, suggesting CREB was a regulator of FNDC5 expression.Functionally,irisin treatment enhanced mitochondrial biogenesis of C2C12 myotubes through increasing peroxisome proliferatoractivated receptor gamma coactivator1 α (PGC1 α), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) expressions, leading to increase myotube mitochondrial respirations and ATP production.Conclusions Our observation indicates that irisin is a metabolic modulator of skeletal muscle, whose expression is controlled by cAMP pathway and intracellular level of calcium.