The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1α protein that acts as a dominant-negative HIF-1α (HIF-1α-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1α was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1α-no-TAD or nontoxic concentrations (0.1 μM; <IC₁₀) of KW2152 and DX-52-1 and exposed to etoposide in air or anoxia (<0.01% oxygen). Topotecan inhibited HIF-1 activity only at cytotoxic concentrations and was not used in the combination study. Etoposide IC₅₀ values in anoxia were 3-fold higher than those in air for HT1080 (2.2 ± 0.3 versus 0.7 ± 0.2 μM) and HCT116 (9 ± 4 versus 3 ± 2 μM) cells. KW2152 and DX-52-1 significantly reduced the anoxic etoposide IC₅₀ in HT1080 cells, whereas only KW2152 yielded sensitization in HCT116 cells. In contrast, AdHIF-1α-no-TAD (multiplicity of infection 50) ablated the anoxic resistance in both cell lines (IC₅₀ values: HT1080, 0.7 ± 0.04 μM; HCT116, 3 ± 1 μM). HIF-1α-no-TAD expression inhibited HIF-1-mediated down-regulation of the proapoptotic protein Bid under anoxia. These data support the potential development of HIF-1 targeted approaches in combination with chemotherapy, where hypoxic cell resistance contributes to treatment failure.