Abstract: Objective To investigate the effect of chloroquine in inducing apoptosis of human hepatocellular carcinoma cells and explore the possible mechanism. Methods MTT assay and flow cytometry were used to evaluate chloroquine-induced growth inhibition and apoptosis in human hepatocellular carcinoma HepG2 cells, respectively. The ATP levels in chloroquinetreated cells were detected using an ATP assay kit. PCR and Western blotting were used to detect the expression levels of miR- 26b and Mcl-1 in the cells, respectively. Results Chloroquine inhibited the proliferation of HepG2 cells in a time- and concentration-dependent manner. Treatments with 80 μmol/L chloroquine for 24, 48, and 72 h induced survival rates of (71.59± 0.2)%, (45.40±0.5)%, and (26.34±1.4)% in the cells. Treatments with chloroquine at 40, 80, and 160 μmol/L for 5 h resulted in obviously lowered intracellular ATP levels in the cells to 87.80%, 71.29%, and 38.02% of the control level, respectively. At 80 μmol/L, chloroquine significantly increased the expression of miR-26b and down-regulated the expression of Mcl-1 in HepG2 cells, and the application of the miR-26b inhibitor increased the cellular expression of Mcl-1. Conclusions Chloroquine can inhibit the cell proliferation, reduce ATP level and induce apoptosis in HepG2 cells possibly through miR-26b-mediated regulation of Mcl-1.