Enterovirus 71 (EV71) is the major causative agent of severe hand, foot, and mouth disease, which affects millions of young children in the Asia-Pacific region annually. In this study, we engineered a novel EV71 virus-like particle (VLP) that lacks VP4 (therefore designated VLP.VP4) and investigated its structure, antigenicity, and vaccine potential. The cryo-electron microscopy (cryo-EM) structure of VLP.VP4 was reconstructed to 3.71-A resolution. Results from structural and biochemical analyses revealed that VLP.VP4 resembles the end product of the viral uncoating process, the 80S empty capsid. VLP.VP4 is able to elicit high-titer neutralizing antibodies and to fully protect mice against lethal viral challenge. Mechanistic studies showed that, at the cellular level, the anti-VLP.VP4 sera exert neutralization effects at both preand postattachment stages by inhibiting both virus attachment and internalization, and at the molecular level, the antisera can block multiple interactions between EV71 and its key receptors. Our study gives a better understanding of EV71 capsid assembly and provides important information for the design and development of new-generation vaccines for EV71, and perhaps for other enteroviruses, as well. IMPORTANCE Enterovirus 71 (EV71) infection may lead to severe hand, foot, and mouth disease, with significant morbidity and mortality. Knowledge regarding EV71 particle assembly remains limited. Here, we report the generation and characterization of a novel EV71 virus-like particle that lacks the VP4 capsid subunit protein. This particle, termed VLP.VP4, structurally mimics the 80S empty capsid, which is the end stage of EV71 uncoating. We further show that VLP.VP4 exhibits desirable immunogenicity and protective efficacy in proof-of-concept studies. In addition, the inhibitory mechanisms of the VLP.VP4-induced antibodies are unraveled at both the cellular and molecular levels. Our work provides the first evidence of picornaviral particle assembly in the complete absence of VP4 and identifies VLP.VP4 as an improved EV71 vaccine candidate with desirable traits. These findings not only enhance our understanding of particle assembly and uncoating of picornaviruses, but also provide important information for structure-guided vaccine design for EV71 and other enteroviruses.