Objective To investigate the effect of hydroxysafflor yellow A (HSYA) on oxidative stress and apoptosis of H9C2 cardiomyocytes after oxygen-glucose deprivation/ reperfusion injury and to explore its possible mechanism. Methods After rat H9C2 cardiomyocytes were stably subcultured to the third passage, they were randomly divided into control, hypoxia/ reoxygenation model (OGD/ R), low-dose HSYA (40 μmol/ L; HSYA40), and high-dose HSYA (80 μmol/ L; HSYA80) groups with 10 duplicate wells in each group. After 6 hours of treatment, the cells were analyzed by CCK-8 assays and aspartate transferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) activities in the medium were measured by a biochemical analyzer. Corresponding kits were used to measure the contents of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in cells. Flow cytometry was used to assess the apoptosis rate. Western blot was used to detect apoptosis-related proteins B-cell lymphoma/ leukemia-2 (Bcl2) and Bcl2-related X Protein (Bax) in cells. Caspase-3 expression was analyzed semiquantitatively. Results Compared with the normal group, cell proliferation in the OGD/ R group was significantly lower, AST, CK, and LDH activities in the medium were significantly increased, SOD and CAT activities were significantly decreased, and the MDA content was increased. The apoptosis rate was significantly increased, Bcl2 and Bax mRNA expression was significantly increased, Bcl2/ Bax protein expression was significantly decreased, and Caspase-3 protein expression was significantly increased. Compared with the OGD/ R group, cell proliferation in the hydroxysafflor A administration group was significantly increased, AST, CK, and LDH activities in the medium were significantly decreased, SOD and CAT activities were significantly increased, and the MDA content was decreased. The apoptosis rate was significantly reduced and expression of anti-apoptotic gene Bcl-2 was upregulated, while the expression of proapoptotic gene Bax was significantly downregulated, Bcl2/ Bax protein expression was significantly increased, and caspase-3 protein expression was significantly decreased. Conclusions HSYA protects H9C2 cardiomyocytes from oxygen-glucose deprivation/ reperfusion injury by inhibiting oxidative stress injury and apoptosis. Its mechanism may be related to improving the activity of antioxidant enzymes, upregulating Bcl-2 gene expression, downregulating Bax gene expression, increasing Bcl-2/ Bax protein expression, and inhibiting the expression of pro-apoptotic protein Caspase-3.