4-O-Methylascochlorin (MAC), a methylated derivative of ascochlorin, was previously shown to promote the accumulation of hypoxia-inducible factor (HIF)-1α in human breast adenocarcinoma MCF-7 cells. In the present study, we further investigated the effects of MAC on the expression and function of HIF-1α in human fibrosarcoma HT-1080 cells. MAC promoted the accumulation of the HIF-1α protein without affecting its constitutive mRNA expression and augmented the transcriptional activation of HIF target genes. Ascorbate, but not N-acetylcysteine, attenuated MAC-mediated HIF-1α accumulation. MAC-induced increases in HIF-1α transcriptional activity were also attenuated by ascorbate. MAC inhibited the hydroxylation of HIF-1α at the proline 564 residue, while it was reversed by ascorbate. MAC slightly decreased the intracellular concentration of ascorbate. The present results demonstrated that MAC promoted the accumulation of HIF-1α by preventing prolyl hydroxylation, and ascorbate attenuated the MAC-mediated inhibition of HIF-1α prolyl hydroxylation.