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Coxsackievirus A5 (CV-A5) has recently emerged as a main hand, foot, and mouth disease (HFMD) pathogen. Following a large-scale vaccination campaign against enterovirus 71 (EV-71) in China, the number of HFMD-associated cases with EV-71 was reduced, especially severe and fatal cases. However, the total number of HFMD cases remains high, as HFMD is also caused by other enterovirus serotypes. A multivalent HFMD vaccine containing 4 or 6 antigens of enterovirus serotypes is urgently needed. A formaldehyde-inactivated CV-A5 vaccine derived from Vero cells was used to inoculate newborn Kunming mice on days 3 and 10. The mice were challenged on day 14 with a mouse-adapted CV-A5 strain at a dose that was lethal for 14-day-old suckling mice. Within 14 days postchallenge, groups of mice immunized with three formulations, empty particles (EPs), full particles (FPs), and a mixture of the EP and FP vaccine candidates, all survived, while 100% of the mock-immunized mice died. Neutralizing antibodies (NtAbs) were detected in the sera of immunized mice, and the NtAb levels were correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak or not observed in the immunized mice compared with those in alum-inoculated control mice. Another interesting finding was the identification of CV-A5 dense particles (DPs), facilitating morphogenesis study. These results demonstrated that the Vero cell-adapted CV-A5 strain is a promising vaccine candidate and could be used as a multivalent HFMD vaccine component in the future. [ABSTRACT FROM AUTHOR]