Enterovirus 71 (EV71) is one of the leading causes of hand, foot and mouth disease with neurological complications in some cases. To study the pathogenesis of EV71 infection, large scale analyses of EV71 infected cells have been performed. However, most of these studies employed rhabdomyosarcoma (RD) cells or used transcriptomic strategy. Here, we performed SILAC-based quantitative proteomic analysis of EV71-infected U251 cells, a human glioma cell line. A total of 3,125 host proteins were quantified, 451 of which were differentially regulated as a result of EV71 infection at 8 hpi or 20 hpi or both.