Several reports have shown that thymoquinone (TQ) effectively attenuates angiogenesis in cancer cells, resulting in suppression of tumor growth. However, it is not yet clear whether TQ reduces hypoxia-inducible factor-1α (HIF-1α) expression in hypoxic cancer cells. Here, we found that TQ was a novel HIF-1α inhibitor through hypoxia response element (HRE)-luciferase assay-based large screening by using 502 natural compounds containing chemical library. TQ reduced HIF-1α protein levels in renal cancer cells; however, it did not affect the HIF-1α protein levels in the presence of proteasome inhibitor, MG132, indicating that the reduction effects of TQ on HIF-1α protein are mediated via the ubiquitination-proteasome dependent pathway. TQ boosted HIF-1α protein degradation, and the mechanism was revealed by inhibiting interaction between HSP90 and HIF-1α. TQ suppressed downstream genes of HIF-1α, indicating negative impact of TQ on HIF-1α transcriptional activities. In addition, TQ altered glucose, lactate, and ATP levels, leading to anaerobic metabolic disturbance. TQ induced apoptosis in hypoxic cancer cells as determined by crystal violet staining and flow cytometry for annexin V-stained cells. Taken together, we suggested that TQ is a potential anticancer agent targeting HIF-1α.