BACKGROUND:: Polyurethane as a popular medical polymer material plays a crucial role in many artificial organs and medical devices. OBJECTIVE:: To prepare nano-silver-zirconium phosphate antibacterial polyurethane and to observe its cytotoxic effect on L929 cells. METHODS:: L929 cells were incubated into 96-well plates and divided into three groups: experimental group treated with antibacterial polyurethane, positive control group with phenol solution, negative control group with high-density polyethylene extract. The cell morphology was observed under inverted phase contrast microscope after 24, 48 and 72 hours. The relative growth rate (RGR) was calculated and the cytotoxicity was graded by with MTT method. RESULTS AND CONCLUSION:: After cultured for 24, 48 and 72 hours, the RGR of L929 cells in the experimental group was 94.3%-97.9%, 94.5%-99.8% and 90.8%-96.3%, respectively. The cytotoxicity of the antibacterial polyurethanes was graded level I. Antibacterial polyurethane has no cytotoxicity to L929 cells and shows good biocompatibility, meeting the safety standards of biomedical materials.