Objective To explore the mechanism by which enterovirus 71 (EV71) crossing the blood-brain barrier (BBB). Methods BBB models were established by human brain microvascular endothelial cells (HBMECs) in vitro and were randomly assigned to control and EV71-infected groups. The cytotoxic effect of EV71 on HBMECs was examined by morphological observation, immunoflurescence, transmission electron microscope and real-time PCR. In addition, the cytoskeletal alterations in HBMECs were observed by laser confocal method. Results EV71 infection could lead to cytopathic changes of HBMECs. Immunofluorescent staining confirmed the presence of EV71 antigen (red fluorescence) and 20-30 nm EV71 particles in EV71-infected HBMECs, but not in mock infected cells. Viral replication in HBMECs was shown by real-time PCR in a time-dependent manner (P<0.01). EV71 infected cell lost the normal morphology and polarity, with disarranged actin filaments. Conclusion For the first time we prove that EV71 can infect HBMECs and can replicate in the cells, and can induce cytoskeleton changes in some HBMECs.