Human Enterovirus 71 (EV71) is recognized as the leading causative agent of hand-foot-and-mouth disease (HFMD) in the Asia-Pacific region in recent years. There are still no approved antiviral drugs or vaccines against EV71 infection yet. In this study, we have developed an advanced platform for production of the virus-like particles (VLPs) for EV71 in Saccharomyces Cerevisiae by co-expressing P1 and 3CD genes of EV71. These VLPs exhibited similar morphology and protein composition as EV71 empty particles produced from EV71-infected cells. Immunization with VLPs in mice elicited robust neutralization antibodies against EV71 and potent cellular immune response. In vivo challenge experiments showed that the immune sera induced by VLP conferred protection in neonate mice against lethal EV71 challenge. Together, our study indicated that VLP from yeast is another potential vaccine candidate against EV71 infection. Copyright © 2013 Elsevier Ltd. All rights reserved.