Hypoxia may activate survival signals in cancer cells. Moreover, hypoxic cells are less sensitive than normoxic cells to doxorubicin cytotoxicity, a potent activator of the p53 tumor suppressor gene. N-myc downstream-regulated gene-1 (NDRG1) is a hypoxia- and retinoic acid-inducible protein, and has been previously implicated in carcinogenesis. As this protein is also a downstream target of p53 and hepatocellular carcinoma (HCC) cells frequently evidence resistance to retinoic acid (RA) cytotoxicity, we attempted to determine whether the suppression of NDRG1 expression may sensitize HCC cells to doxorubicin and/or RA cytotoxicity. HCC cells expressed NDRG1 protein, and the expression of this protein was hypoxia- and RA-inducible. Doxorubicin treatment induced HCC cell cytotoxicity via the activation of mitochondrial apoptotic signals, including caspase-9 activation. Hypoxic HCC cells are less sensitive to doxorubicin-induced apoptosis. The suppression of NDRG1 expression either by siRNA or flavopiridol sensitized hypoxic HCC cells to doxorubicin cytotoxicity, and this was attributed to more profound augmentation of JNK and caspase-9 activation. The suppression of NDRG1 expression also sensitized RA-resistant HCC cells to RA-induced apoptosis, and this sensitization was more apparent in hypoxic HCC cells than in normoxic cells. Glutaredoxin2 expression was down-regulated in NDRG1-suppressed HCC cells. These results show that hypoxia- and RA-inducible NDRG1 expression is responsible for doxorubicin and RA resistance in HCC cells. Thus, the selective interruption of NDRG1 signaling may prove to be therapeutically useful in HCCs, particularly in the advanced infiltrative type of tumors exposed to hypoxic environments.