Objective It is difficult to treat chronic osteomyel itis due to the formation of the Staphylococcus aureus biofilms. Liposomal gentamicin-impregnated allogeneic cortical bone can inhibit the formation of the Staphylococcus aureusbiofilms. To explore the treatment of chronic osteomyel itis of rabbit by l iposomal gentamicin-impregnated allogeneic cortical bone. Methods The l iposomal gentamicin, l iposomal gentamicin-impregnated allogeneic cortical bone and gentamicinimpregnated allogeneic cortical bone were produced. Then the chronic Staphylococcus aureus osteomyel itis models of rabbit were made in left lower l imbs of 40 6-month-old rabbits and the right lower l imbs were used as controls. After 2 weeks, the observations of gross and X-ray were done. Four rabbits died within 10 days after the models were made and other 36 rabbits were devided into 6 groups: group A (no antibiotics), group B (intravenous injection of gentamicin), group C (intravenous injection of l i posomal gentamicin), group D (implantation of gentamicin-impregnated allogeneic cortical bone), group E (implantation of l i posomal gentamicin-impregnated allogeneic cortical bone), and group F (implantation of allogeneic cortical bone). After 2 weeks of treatment, the bacterial culture, X-ray and HE staining were done. Results The chronic Staphylococcus aureus osteomyel itis model of rabbit was made successfully. The X-ray showed dissolution of bone and periosteal reaction in groups A, B, C, and F, and no obvious dissolution of bone and periosteal reaction in groups D and E. The Norden scores were (2.5 ± 0.3), (2.1 ± 0.2), (1.5 ± 0.3), (1.5 ± 0.2), (0.9 ± 0.3), and (2.7 ± 0.3) points in groups A-F, respectively; showing significant differences between group A and groups B-E (P lt; 0.05), between groups B, E, F and other groups (P lt; 0.05). The results of blood and marrow cultures for Staphylococcus aureus were positive in groups A and F, and negative in other 4 groups; the results of bone marrow culture for Staphylococcus aureus were positive in 6 rabbits of group B, 4 rabbits of group C and 3 rabitts of group D; and the results were negative in group E. HE staining showed: in groups A and F, abscess and dead bone formed, and no new bone formation were observed; in groups B and C, different degrees of neutrophil accumulation was seen; in group D, some neutrophil accumulation occurred, and osteoprogenitor cells and osteoclasts were seen around implanted bone; and in group E, no neutrophil accumulation was observed, a lot of granulation tissues formed, and osteoprogenitor cells and osteoclasts were seen around implanted bone. Conclusion Implantation of l iposomal gentamicin-impregnated allogeneic cortical bone has remarkly better effect in treating chronic osteomyel itis than intravenous injection of l iposomal gentamicin and implantation of gentamicin-impregnated allogeneic cortical bone.