Objective Hand, foot and mouth disease ( HFMD) mainly affects children under the age of 5. Because HFMD is caused by a group of enteroviruses, a vaccine targeting only one pathogen would be greatly limited, and a multivalent vaccine would be more effective. In this study, we prepared a trivalent VP1 protein vaccine against EV71, CA16 and CA10 viruses. This vaccine was then injected into mice to examine immune activities and to evaluate safety. Methods After immunization with the trivalent VP1 protein vaccine, virus-specific antibodies and their neutralizing activities were analyzed, and virus-specific T cell immune responses and changes in cytokines were assessed. Furthermore, passive immunoprotective effects passed from immunized mothers to their neonatal offspring were evaluated with in vivo infection experiments. Results The trivalent VP1 protein vaccine continuously induced virus-specific IgGs and IgMs, which had the ability to neutralize the viruses. In addition to inducing virus-specific humoral immunity, the trivalent VP1 protein vaccine activated the virus-specific T cell response as well. The vaccine also provided passive immunity to neonatal mice against EV71 virus infection. Additionally, the stable levels of inflammatory cytokines during the immune response indicated that the vaccine was safe. Conclusions The trivalent VP1 protein vaccine was effective against EV71, CA16 and CA10 viral challenge, and could therefore be useful for the development of multivalent HFMD vaccines.