Circulating enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) is a major public health problem in the Asian-Pacific region. An EV71 vaccine for HFMD prevention is currently being developed. However, viral determinants that could influence the vaccine's efficacy have not been well characterized. In this study, we isolated and characterized several EV71 strains that are currently circulating in northern and southern China. We determined that VP1 variation is a major determinant of EV71 immunogenicity. A single amino acid variation in VP1 can lead to significant differences in the breadth and potency of immune responses against primary EV71 isolates as well as the sensitivity of EV71 to heterologous neutralizing antibody responses. We also identified EV71 strains that could induce potent immunogenic and cross-neutralizing antibody responses against diverse EV71 strains. Furthermore, these neutralizing antibodies could protect neonatal mice from lethal dose challenge with various circulating EV71 viruses. Our study provides useful information for EV71 vaccine development and evaluation.