Hypoxia-inducible transcription factors 1 and 2 (HIFs) are major mediators of cancer development and progression and validated targets for cancer therapy. Although calcitriol, the biologically active metabolite of vitamin D, was attributed with anticancer properties, there is little information on the effect of calcitriol on HIFs and the mechanism underling this activity. Here, we demonstrate the negative effect of calcitriol on HIF-1/2α protein levels and HIF-1/2 transcriptional activity and elucidate the molecular mechanism of calcitriol action. We also reveal that the suppression of vitamin D receptor (VDR) expression by siRNA does not abrogate the negative regulation of HIF-1α and HIF-2α protein levels and HIF-1/2 transcriptional activity by calcitriol, thus testifying that the mechanism of these actions is VDR independent. At the same time, calcitriol significantly reduces the phosphorylation of Akt protein kinase and its downstream targets and suppresses HIF-1/2α protein synthesis by inhibiting HIF1A and EPAS1 (Endothelial PAS domain-containing protein 1) mRNA translation, without affecting their mRNA levels. On the basis of the acquired data, it can be proposed that calcitriol reduces HIF-1α and HIF-2α protein levels and inhibits HIF-1 and HIF-2 transcriptional activity by a VDR-independent, nongenomic mechanism that involves inhibition of PI3K/Akt signaling pathway and suppression of HIF1A and EPAS1 mRNA translation.