Abstract: In present study, we investigated the mechanism of regulating HIF-1α expression by hydroxysafflor yellow A (HSYA)in Eahy 926 cell line under 1% O₂ hypoxia. Eahy 926 cells were incubated with HSYA (100, 10 and 1 μmol·L^-1) under hypoxia for the indicated time after treatment. Cell proliferation rate was detected using MTT assays. VHL and p53 location and protein expression were analyzed by immunocytochemical stain. HIF-1α, VHL and p53 mRNA expression were detected by RT-PCR. Protein expression of HIF-1α, VHL and p53 were assayed by Western blotting method. HSYA at 100 μmol·L^-1 increased Eahy 926 cells proliferation rate under hypoxia. HIF-1α mRNA and protein expression were up-regulated in the presence of HSYA. VHL, p53 mRNA and protein expression decreased significantly after 8 hours of treatment under hypoxia. HSYA protected Eahy 926 cells from hypoxia, and up-regulated HIF-1α expression partially via its inhibition of VHL and p53 expression.