Aminoflavone (AF), the active component of a novel anticancer agent (AFP464) in phase I clinical trials, is a ligand of the aryl hydrocarbon receptor (AhR). AhR dimerizes with HIF-1beta/AhR, which is shared with HIF-1alpha, a transcription factor critical for the response of cells to oxygen deprivation. To address whether pharmacologic activation of the AhR pathway might be a potential mechanism for inhibition of HIF-1, we tested the effects of AF on HIF-1 expression. AF inhibited HIF-1alpha transcriptional activity and protein accumulation in MCF-7 cells. However, inhibition of HIF-1alpha by AF was independent from a functional AhR pathway. Indeed, AF inhibited HIF-1alpha expression in Ah(R100) cells, in which the AhR pathway is functionally impaired, yet did not induce cytotoxicity, providing evidence that these effects are mediated by distinct signaling pathways. Moreover, AF was inactive in MDA-MB-231 cells, yet inhibited HIF-1alpha in MDA-MB-231 cells transfected with the SULT1A1 gene. AF inhibited HIF-1alpha mRNA expression by approximately 50%. Notably, actinomycin-D completely abrogated the ability of AF to downregulate HIF-1alpha mRNA, indicating that active transcription was required for the inhibition of HIF-1alpha expression. Finally, AF inhibited HIF-1alpha protein accumulation and the expression of HIF-1 target genes in MCF-7 xenografts. These results show that AF inhibits HIF-1alpha in an AhR-independent fashion, and they unveil additional activities of AF that may be relevant for its further clinical development.