Chemoresistance in hepatocellular carcinoma (HCC) is associated with multiple cellular responses to environmental stresses, such as nutrient deprivation and hypoxia. Nevertheless, whether ER stress resulting from nutrient deprivation and tumor hypoxia contributes to drug resistance remains unclear. Melatonin increased the efficacy of chemotherapeutic drugs in hepatocellular carcinoma in our previous studies. However, the effects of melatonin on endoplasmic reticulum (ER) stress-induced resistance to chemotherapeutic agents in HCC have not been tested. The effect of the endoplasmic reticulum (ER) stress response during resistance of human hepatocellular carcinoma cells against doxorubicin was investigated in this study. Pretreatment of HepG2 and SMMC-7721 cells (two human hepatocellular carcinoma cell lines) with tunicamycin, an ER stress inducer, drastically decreased the rate of apoptosis generated by doxorubicin. Interestingly, co-pretreatment with tunicamycin and melatonin significantly increased apoptosis induced by doxorubicin. Simultaneously, the expression of phosphorylated AKT (p-AKT) was elevated in HepG2 and SMMC-7721 cells given tunicamycin but reduced in the presence of melatonin. Furthermore, consistent with inhibition of AKT activation by using the PI3K inhibitor LY294002, melatonin elevated the levels of CHOP (C/EBP-homologous protein) and reduced the levels of Survivin (a member of the inhibitor of apoptosis protein family)suggesting that inhibition of the PI3K/AKT pathway by melatonin-reversed ER stress-induced resistance to doxorubicin in human hepatocellular carcinoma cells. These results demonstrate that melatonin attenuates ER stress-induced resistance to doxorubicin in human hepatocellular carcinoma cells by down-regulating the PI3K/AKT pathway, increasing the levels of CHOP and decreasing the levels of Survivin.