Aims This work explored the effects of irisin on metabolism, gene expression and mitochondrial content in cultured myocytes. Methods C2C12 myocytes were treated with various concentrations of irisin for various durations. Glycolysis and oxidative metabolism were quantified by measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and mitochondrial content was assessed by flow cytometry and confocal microscopy. Results Cells treated with irisin exhibited significantly increased oxidative metabolism. Irisin treatment also significantly increased mitochondrial uncoupling at various doses and durations. Lastly, treatment with irisin also significantly elevated metabolic gene expression including peroxisome proliferator‐activated receptor γ coactivator‐1 alpha (PGC‐1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), irisin, glucose transporter 4 (GLUT4) and mitochondrial uncoupling protein 3 (UCP3) leading to increased mitochondrial biogenesis. Conclusions Our observations are the first to document increased metabolism in myocytes through irisin‐mediated induction of mitochondrial biogenesis and uncoupling with corresponding gene expression. These observations support the need for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin‐based therapy.