AIM: To investigate the role of ribonucleotide reductase M2 (RRM2) in 5-fluorouracil (5-FU) resistance of HCC cells and to develop potential strategies to enhance the sensitivity of HCC cells to 5-FU. METHODS: The expression of RRM2 was examined by Western blot in BEL/5-FU cells and BEL7402 cells. The expression of RRM2 was down-regulated through RNA interference (RNAi) technology and the activity of RRM2 was inhibited by the RRM2 inhibitor 3-AP. The cell proliferation ability was detected by CCK-8 assay and colony formation assay, and the apoptosis was analyzed by Confocal high-content system. RESULTS: The expression level of RRM2 was increased by 2.5-fold in BEL/5-FU cells compared with BEL7402 cells. Compared with control siRNA, the half maximum inhibitory concentration IC