Abstract In this study, a new microcomposite composed of nickel ion and chitosan was prepared for the purpose of inhibiting enterovirus 71 (EV71) infections. A Ni-chitosan (NiCS) microcomposite and a chitosan microcomposite (CS) were applied to treat Vero cells either during or after EV71 virus adsorption. During a 72-h period of post-infection, the addition of the NiCS microcomposite during virus adsorption exhibited an inhibitory effect on EV71 infection. An excellent effect of over 100% average relative cell viability was obtained and no infection occurred when ≥300μl of NiCS microcomposite was added. However, the addition of NiCS microcomposite after virus adsorption revealed a reduced inhibitory effect. Conversely, the cells treated with CS microcomposite showed a high rate of cell death caused by EV71 infection. The inhibitory effects of NiCS microcomposite on EV71 infection revealed no appearance of CPE in the cells and no viral particle synthesis, and the presence of nickel ion bound to the VP1 protein of EV71 prevented the entry and uncoating of EV71. Our results indicate the potential inhibitory effects of NiCS microcomposite on enterovirus 71 infections.