Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, is resistant to anticancer drugs. Hypoxia is a major cause of tumor resistance to chemotherapy, and hypoxia-inducible factor (HIF)-1 is a key transcription factor in hypoxic responses. We have previously demonstrated that gene transfer of an antisense HIF-1alpha expression vector downregulates expression of HIF-1alpha and vascular endothelial growth factor (VEGF), and synergizes with immunotherapy to eradicate lymphomas. The aim of the present study was to determine whether gene transfer of antisense HIF-1alpha could enhance the therapeutic efficacy of doxorubicin to combat HCC. Both antisense HIF-1alpha therapy and doxorubicin suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, tumor angiogenesis, and cell proliferation, and induced tumor cell apoptosis. The combination therapy with antisense HIF-1alpha and doxorubicin was more effective in suppressing tumor growth, angiogenesis, and cell proliferation, and inducing cell apoptosis than the respective monotherapies. Gene transfer of antisense HIF-1alpha downregulated the expression of both HIF-1alpha and VEGF, whereas doxorubicin only downregulated VEGF expression. Antisense HIF-1alpha and doxorubicin synergized to downregulate VEGF expression. Both antisense HIF-1alpha and doxorubicin inhibited expression of proliferating cell nuclear antigen, and combined to exert even stronger inhibition of proliferating cell nuclear antigen expression. Antisense HIF-1alpha therapy warrants investigation as a therapeutic strategy to enhance the efficacy of doxorubicin for treating HCC.