Objective: To investigate the relationship between the expression of ribosomal S6 kinase 2 (RSK2) and chemoresistance in hepatocellular carcinoma cells (HCC). Methods: Human HCC HepG2 cells were divided into RSK2 interference group (cells were transfected with PGCsi3.0-RSK2 siRNA plasmid), 5-FU treatment group (cells were treated with 5-FU for 24 h), 5-FU treatment+RSK2 interference group and blank control group. The cell proliferation of each group was determined by MTT assay and the protein expression levels of RSK2, c-Jun, Bcl-2 and LKB1 were detected by Western blot analysis. Results: Compared with blank control group, the RSK2 interference and 5-FU treatment group both significantly inhibited the proliferation of HepG2 cells(both P<0.05). Moreover, the combined effect of RSK2 interference and 5-FU treatment was greater than each alone (both P<0.05). Compared with RSK2 interference group, the protein expression levels of RSK2, c-Jun and Bcl-2 were increased and LKB1 protein level was decreased in cells of 5-FU treatment+RSK2 interference group (all P<0.05). Conclusion: Inhibition of RSK2 expression can enhance the sensitivity of HCC to chemotherapeutic agent 5-FU, and the mechanism may be related to its regulating the expression of c-Jun, Bcl-2 and LKB1.