Objective To study the mechanism ofhuman enterovirus 71 (EV71) entering human neuroblastoma SK-N-SH cells. Methods After the SK-N-SH cells were pretreated with chlorpromazine (CPZ) or nystatin (NT), real-time quantitative PCR (qRT-PCR) was employed to measure EV71 mRNA level, and indirect immunofluorescence microscopy was used to detect the expression level of viral protein 1 (VP1) in the target cells. In order to reveal the colocalization of EV71 with clathrin, laser confocal microscopy was performed on the infected cells. Results CPZ could significantly inhibit EV71 mRNA level and the expression of VP1 in the target cells, while NT had no effect on EV71 infection. Confocal microscopy showed that EV71 was colocalize with clathrin. Conclusion EV71 infects human neuroblastoma SK-N-SH cells by the clathrin-mediated endocytosis.