Hydroxysafflor yellow A (HSYA), is a component of the flower, Carthamus tinctorius L. In this study, we investigated its effect on Human Umbilical Vein Endothelial Cells (HUVECs) under hypoxia. We evaluated cell viability using the MTT kit. The cell cycle distribution was analyzed by PI staining flow cytometric analysis. PI AnnexinV-FITC detection and the TUNEL assay were performed to evaluate the apoptosis rate. Nitric oxide (NO) generation in cell supernatant was measured by the Griess assay. RT-PCR, Western blot and Immunocytochemistry analysis were used to evaluate the changes of Bcl-2, Bax, p53 and eNOS. Our data showed that HSYA inhibited cell apoptosis and cell cycle G1 arrest induced by hypoxia. HSYA treatment increased the Bcl-2/Bax ratio of protein and mRNA, reduced p53 protein expression in cell nucleus. In addition, HSYA enhanced the NO content of cell supernatant under hypoxia, accompanied with upregulating eNOS mRNA expression and protein level. Taken together, these results demonstrate that HSYA could protect HUVECs from hypoxia induced injuries by inhibiting cell apoptosis and cell cycle arrest. These findings have partly revealed the molecular mechanism of HSYA on treating of ischemic heart disease. We expected our experiments might provide some clues for further research.