We have previously reported on the inhibition of HIF-1α (hypoxia-inducible factor α)-regulated pathways by HEXIM1 [HMBA (hexamethylene-bis-acetamide)-inducible protein 1]. Disruption of HEXIM1 activity in a knock-in mouse model expressing a mutant HEXIM1 protein resulted in increased susceptibility to the development of mammary tumours, partly by up-regulation of VEGF (vascular endothelial growth factor) expression, HIF-1α expression and aberrant vascularization. We now report on the mechanistic basis for HEXIM1 regulation of HIF-1α. We observed direct interaction between HIF-1α and HEXIM1, and HEXIM1 up-regulated hydroxylation of HIF-1α, resulting in the induction of the interaction of HIF-1α with pVHL (von Hippel–Lindau protein) and ubiquitination of HIF-1α. The up-regulation of hydroxylation involves HEXIM1-mediated induction of PHD3 (prolyl hydroxylase 3) expression and interaction of PHD3 with HIF-1α. Acetylation of HIF-1α has been proposed to result in increased interaction of HIF-1α with pVHL and induced pVHL-mediated ubiquitination, which leads to the proteasomal degradation of HIF-1α. HEXIM1 also attenuated the interaction of HIF-1α with HDAC1 (histone deacetylase 1), resulting in acetylation of HIF-1α. The consequence of HEXIM1 down-regulation of HIF-1α protein expression is attenuated expression of HIF-1α target genes in addition to VEGF and inhibition of HIF-1α-regulated cell invasion.