Hesperetin, a flavonoid from citrus fruits, has been proved to possess biological activity on various types of human cancers. However, few related studies on hepatocellular carcinoma are available. In this study, we aimed to investigate the effect of hesperetin on hepatocellular carcinoma cells in vitro and in vivo and clarify its potentially specific mechanism. Compared with the control group, the proliferations of hepatocellular carcinoma cells in hesperetin groups were significantly inhibited (P < 0.05), and a dose- and time-dependent inhibition of cell viability was observed. When pretreated with H2O2 (1 mM) or N-acetyl-L-cysteine (5 mM), the inhibition of cell viability by hesperetin was enhanced or reduced, respectively (P < 0.05). Similarly, the levels of intracellular ROS, ATP and Ca(2+) changed in different groups (P < 0.05). The results of Hoechst 33258 staining showed that the percentages of apoptotic cells in hesperetin groups are remarkably higher than that in control group (P < 0.05). And the results of Western blot showed that hesperetin caused an increase in the levels of cytosolic AIF, cytosolic Apaf-1, cytosolic Cyt C, caspase-3, caspase-9 and Bax and a decrease in that of Bcl-2, mitochondrial AIF, mitochondrial Apaf-1 and mitochondrial Cyt C (P < 0.05). Meanwhile, hesperetin significantly inhibited the growth of xenograft tumors. Our study suggests that hesperetin could inhibit the proliferation and induce the apoptosis of hepatocellular carcinoma via triggering the activation of the mitochondrial pathway by increasing the levels of intracellular ROS, ATP and Ca(2+).