Abstract: Objective：To investigate the role of c-Jun N-terminal kinase (JNK1/2) signaling pathway in enterovirus 71 (EV71) infection. Methods：The effects of different concentrations of SP600125 on the activity of human rhabdosarcoma (RD) cells were detected by trypanbalu staining. The levels of VP1 mRNA and protein in EV71infected RD cells were detected by real time Q-PCR and western blot, respectively. The levels of total and phosphorylated JNK1/2, c-Fos and c-Jun protein were determined by western blot. Last, the effects of JNK1/2 inhibitor SP600125 on EV71 replication and JNK1/2 signaling pathway were analyzed. Results：The results of trypanbalu staining showed that 5 and 10 μmol/L of SP600125 didn′t influence on the activity of RD cells (P>0.05), while 20 μmol/L of SP600125 decreased the survival of RD cells significantly (P<0.05). Compared with the control, the expression levels of VP1 mRNA and protein in EV71-infected RD cells decreased obviously at 8 hours postinfection (P<0.01). In addition, after RD cells were infected EV71, the levels of phosphorylated JNK1/2, c-Fos and c-Jun increased significantly (P<0.05). However, the pretreatment of SP600125 decreased the phosphorylation levels of JNK1/2, c-Fos and c-Jun protein obviously (P<0.05). Conclusion：EV71 infection may effectively activate the JNK1/2 signaling pathway in RD cells, which may be related to EV71 replication.