Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease (HFMD), which mainly occurs in children. Children with EV71 infection can develop severe neurological diseases. Heat shock protein 78 (HSP78) facilitates proper protein folding during viral propagation and is induced during virus infection. Nevertheless, the role that HSP78 plays during EV71 infection is still unclear. In this study, recombinant HSP78 protein was expressed in a prokaryotic expression system and used for exploring the interaction between HSP78 and EV71 propagation. Detection using a mouse immune anti-HSP78 serum in ELISA and western blot demonstrated that the recombinant HSP78 antigen is highly immunogenic. Furthermore, the recombinant HSP78 protein was able to bind to EV71 VP1 and intensified the cytopathic effect and viral propagation during EV71 infection, while the immune serum had a counteractive effect. However, knockdown of the HSP78 gene in Vero cells before EV71 infection did not result in a reduced virus titer. In addition, HSP78 on the cell surface was upregulated in human neuroblastoma cells (SK-N-SH) infected with EV71.