Objective To establish and evaluate an animal model of chronic osteomyelitis of femur in SD rats. Methods Twenty-four male SD rats were used to construct the osteomyelitis model. The intercondylar groove of the femur was exposed in rats, and a small hole with diameter of 0.5 mm was drilled by electric drill into the intercondylar groove of femur through the femoral bone marrow cavity. Staphylococcus aureus suspension of 50 μL with concentration of 1×10⁶/mL was injected into the right femoral bone marrow cavity of rats, and then the hole was sealed with sterile bone wax. The behavior, wound healing, body temperature, healing of the incision, and gross infection of the lesion were observed after operation. At 2, 4 and 8 weeks after operation, the soft tissue around the femur of rats was taken for bacterial culture, and micro-CT findings of the molded femur were observed. Histopathological sections were analyzed by hematoxylin-eosin, Giemsa and Masson staining at 2 and 8 weeks after surgery. Results?The right lower limb of the osteomyelitis rats was suspended without weight bearing after operation, and the surgical incision completely healed about 10 d later. The animal returned to normal activity level on day 12, and 15 d after surgery the activity level decreased slightly. The body temperature of the rats began to rise after operation, and local redness and swelling occurred. After 2 weeks, the body temperature gradually decreased, but still higher than normal. The local swelling of right knee joint appeared in some rats. After 4 weeks, local swelling and body temperature increased in all the osteomyelitis rats. Staphylococcus aureus colonies were evenly distributed in the soft tissue culture around the femur of rats, and increased with time. Micro-CT results showed that 8 weeks after operation, inflammation reaction was obvious in medullary cavity of rats, with bone fragmentation involving all layers of bone cortex, also with dead bone formation and new bone formation. The histopathological analysis of the femur showed many inflammatory cells infiltrating into the bone marrow cavity with inflammation limited to the bone marrow cavity 2 weeks after operation, the collagen fibers in the cortical bone were arranged continuously, and a large number of Staphylococcus aureus were contained in the tissue. Eight weeks after operation, inflammation persisted, the collagen fibers in the cortical bone were broken and the cortical bone was destroyed, sinus channels were connected with the outside in cortical bone, and the number of Staphylococcus aureus in the tissue increased. Conclusion A model of chronic osteomyelitis of femur has been successfully established in SD rats, which can simulate the pathophysiological change of clinical osteomyelitis and has high replicability.