Objective: To investigate the effect of gentamicin loaded chitosan nanospheres(GS/CS NPs) on the treatment of chronic osteomyelitis of rabbit. Methods: Forty-eight rabbits were used to establish the chronic osteomyelitis model,and observation of affected limb and X-ray were done 3 weeks later. Forty-four rabbits had symptoms and signs of chronic osteomyelitis and were divided into 4 groups. Group A has no treatment,group B was treated with chitosan nanospheres,group C was treated with gentamicin sulfate,and group D was treated with GS/CS NPs. The X-Ray,observation of the bone marrow cavity,bacterial culture and the HE staining were done after 3 weeks of treatment. Results: After 3 weeks of treatment,the X-Ray of groups A~C showed apparent signs of chronic osteomyelitis,the formation of abscess were observed in bone marrow cavity,and the cultures of the Staphylococcus aureus were positive. The HE staining of groups A~C showed neutrophil accumulation,and the formation of dead bone were observed in groups A~B. The Norden and Smeltzer scores showed significant differences between group D and groups A~C(both P < 0.05). Conclusion: GS/CS NPs has significant effects on the treatment of rabbit chronic osteomyelitis as local drug delivery system.