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As a primary active ingredient of safflor yellow, hydroxysafflor yellow A (HSYA) exhibits notable antioxidative and neuroprotective effects. The aim of the present study was to investigate the protective effects of HSYA in mesenchymal stem cells (MSCs) exposed to hypoxia (5% O2) and serum deprivation (H/SD), and to explore the mechanisms underlying HSYA-mediated protection. Under H/SD conditions, HSYA was applied to protect MSCs against injury. Cell viability, proliferation, apoptosis and reactive oxygen species (ROS) levels were determined using an 5-ethynyl-2'-deoxyuridine assay, MTT assay, Hoechst 33342/propidium iodide and 2',7'-dichlorodihydrofluorescein diacetate staining, respectively. The results revealed that 160 mg/l HSYA significantly reduced apoptosis and ROS levels compared with the H/SD group; however, HSYA demonstrated minimal effects on cell proliferation. A western blot assay demonstrated that HSYA reduced cleaved caspase-3 expression and cytC release from the mitochondria to the cytoplasm when compared with the H/SD group. In addition, western blotting and RT-qPCR analyses revealed that HSYA treatment significantly increased the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). In conclusion, the results of the current study demonstrated that HSYA exerts protective effects against H/SD-induced apoptosis in MSCs potentially via activation of the HIF-1α/VEGF signaling pathway and stabilization of the mitochondrial membrane. [ABSTRACT FROM AUTHOR]