The objectives of this work were to develop an antibiotic coating on the surface of a titanium plate to determine its antibacterial properties in vitro and in vivo. To prepare vancomycin-coated titanium implants, we adopted the electrospinning nanotechnique. The surface structure of the coating implants was observed using a scanning electron microscope. An elution method and a high-pressure liquid chromatography assay were used to characterize the release behavior of vancomycin from the coating. The antibacterial efficacy and the cytotoxicity of the coated titanium implants on osteoblasts were investigated in vitro. In addition, X-ray, white blood cell count, C-reactive protein, erythrocyte sedimentation rate, and pathological examination were performed to validate its antimicrobial efficacy in vivo. The antibiotic coating released 82.7% (approximately 528.2 mu g) of total vancomycin loading in the coating in vitro. The release behavior of vancomycin from nanofiber coatings exhibited a biphasic release pattern with an initial burst on day 1, followed by a slow and controlled release over 28 days. There was no cytotoxicity observed in vitro for the vancomycin-loaded coating. The vancomycin-coated titanium implants were active in treating implant-associated infection in vivo. Thus, vancomycin-coated titanium implants may be a promising approach to prevent and treat implant-associated infections.