Titanium and its alloys have been wildly used as bone implants. However, it is still facing a severer issue: implant related infections due to the lack of antibacterial ability. Copper (Cu) has good antibacterial ability and can be used to improve the anti-infection capability of titanium. In this study, three kinds of Ti samples with different contents of Cu in the modified layer were prepared by plasma immersion ion implantation (PIII) technology, and their responses to bacteria and cells were explored in vitro. The results showed that the sample with low Cu content at the surface could promote the proliferation of rat bone marrow mesenchymal stem cells (rBMSCs) and human umbilical vein endothelial cells (HUVECs) but not inhibit the proliferation of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). As the implantation time extends, antibacterial ability of the samples with high Cu content at the surface was significantly enhanced, and no obvious cytotoxicity was observed. Therefore, it is possible to acquire a balance between antibacterial ability and biocompatibility of Ti by controlling the contents of Cu in the modified layer.