Irisin is a polypeptide hormone derived from the proteolytic cleavage of fibronectin-type III domain-containing 5 (FNDC5) protein. Once released to circulation upon exercise or cold exposure, irisin stimulates browning of white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression, leading to an increase in total body energy expenditure by augmented UCP1-mediated thermogenesis. It is currently unknown whether irisin is secreted by bone upon exercise or whether it regulates bone metabolism in vivo. In this study, we found that 2 weeks of voluntary wheel-running exercise induced high levels of FNDC5 messenger RNA as well as FNDC5/irisin protein expression in murine bone tissues. Increased immunoreactivity due to exercise-induced FNDC5/irisin expression was detected in different regions of exercised femoral bones, including growth plate, trabecular bone, cortical bone, articular cartilage, and bone-tendon interface. Exercise also increased expression of osteogenic markers in bone and that of UCP1 in WAT, and led to bodyweight loss. Irisin intraperitoneal (IP) administration resulted in increased trabecular and cortical bone thickness and osteoblasts numbers, and concurrently induced UCP1 expression in subcutaneous WAT. Lentiviral FNDC5 IP administration increased cortical bone thickness. In vitro studies in bone cells revealed irisin increases osteoblastogenesis and mineralization, and inhibits receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis. Taken together, our findings show that voluntary exercise increases irisin production in bone, and that an increase in circulating irisin levels enhances osteogenesis in mice.