Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease associated with neurological complications in young children. Currently, there is no specific treatment for EV-A71 infection due to the inadequate information on viral biology and neuropathogenesis. Among enteroviruses, nonstructural 3A protein mediates the formation of replication organelles which plays a major role in viral RNA synthesis and assembly. Although enteroviral 3A proteins have been intensively studied, the data on EV-A71 3A, especially in neuronal cells, are still limited. In this study, PRSS3 (mesotrypsinogen, also known as brain trypsinogen) was identified as EV-A71 3A-interacting counterpart from the transfected human neuroblastoma SH-SY5Y cells by pull-down assay and liquid chromatography tandem mass spectrometry. It was confirmed that PRSS3 variant 3 derived from human SH-SY5Y cells had the physical interaction with EV-A71 3A. Importantly, the role of PRSS3 in EV-A71 replication was verified by overexpression and siRNA-mediated gene silencing approaches. The detailed mechanism of the PRSS3 involved in EV-A71 replication and neuropathogenesis warrants further experimental elucidation. In conclusion, this study has discovered a novel EV-A71 3A interacting protein that offers the opportunity to study the neuropathogenesis of the infection which paves the way for developing a specific and effective treatment for the disease.