Multidrug resistance is one of the main causes of chemotherapy failure in liver cancer. Liver cancer stem cells (LCSCs) are considered responsible for the recurrence and chemoresistance of liver cancer. Doxorubicin (DOX) is the most frequently used chemotherapeutic agent in the treatment of liver cancer. However, while DOX can kill the bulk liver cancer cells, it fails to eliminate the LCSC population. Activation of autophagy has emerged as a resistance mechanism against DOX in liver cancer cells. However, the association between autophagy and DOX resistance in liver cancer stem cells remains uncertain. We have hypothesized that inhibition of autophagy could sensitize LCSCs to the treatment of DOX. In this study, LCSCs were enriched from liver cancer Huh7 and PLC/PRF/5 cells in non-adherent spheres. The effect of autophagy inhibition on DOX-induced cytotoxicity to LCSCs was evaluated by tumorsphere formation assay. Apoptosis of LCSCs was measured by TUNEL assay and Annexin V assay. The results showed that combined treatment of DOX and autophagy inhibition with either pharmacological inhibitor 3-MA or RNA interference significantly increased the percentage of apoptotic cells compared with DOX treatment. The addition of autophagy inhibition to DOX reduced the frequency of LCSCs compared with DOX treatment indicating the benefit of the combinational therapy. In conclusion, inhibition of autophagy may reverse the resistance of LCSCs to DOX, suggesting autophagy as a potential therapeutic target for liver cancer treatment.