Objective To develop a closed femur fracture model in mice with intramedullary nailing fixation. Methods Twenty femurs of C57BL/6 mice were bisected in order to dentify anatomic characteristics. Totally 50 mice in 12-16 weeks old were used. A 0.2-mm guide wire was inserted into the intramedullary canal through a 0.45-mm stainless injection needle. After a closed diaphyseal fracture by an impact device was produced,a 0.45-mm stainless injection needle was pushed over the guide wire. The guide wire was then removed. The fractures were examined using X-ray and Safranin O- Fast Green stain. Results Thirty-two transverse fractures, 16 oblique fractures and 2 commuted fractures were created. Successful rate of the fracture model with transverse or short oblique fractures was 96%. The X-ray and histological examination confirmed that the fracture healing belonged to typical secondary fracture healing. Conclusion The intramedullary nailing is a safe and convenient method for establishing the closed femur fracture model in mice. The fracture model, which healing is typically endochondral ossification, can be reproducible and used in molecular and genetic study of fracture healing and bone metabolism.