Fracture is the most frequently encountered traumatic large-organ injury observed in human patients. Cordycepin possesses beneficial effects in osteogenesis of mesenchymal stem cells (MSCs), but its effect on fracture healing is largely unknown. A rat model of closed femur fracture was established, and treated with therapy using bone marrow-derived MSCs (BMMSCs). The effect of cordycepin on the osteogenic process of BMMSCs in vitro was evaluated by Alizarin Red S (ARS) staining and expressions of osteogenic marker genes. Radiographic evaluations and four-point bending mechanical testing were performed on model rats after BMMSC treatment, to assess the effect of cordycepin on fracture healing. Cordycepin promoted osteogenesis of BMMSCs in vitro, and enhanced radiographic parameters and mechanical properties in rat closed femur fracture model using BMMSC therapy in vivo. A hypoxia inhibitor echinomycin could negate the above-mentioned therapeutic effects of cordycepin, indicating that the beneficial effects of cordycepin were mediated via hypoxic response pathway. This study demonstrates that cordycepin promotes osteogenesis of BMMSCs and accelerates fracture healing via hypoxia in a rat model of closed femur fracture, and proposes the clinical potential of cordycepin in bone fracture treatments.