Ischemic heart disease is a major health threat, resulting in a large number of mortalities annually worldwide. Oxidative stress is one of the main causes of cell death during ischemia-reperfusion (IR) injury. Cyclin dependent kinase inhibitor 1A (known as p21) is important in protecting tissues against IR injury, however the mechanism remains unknown. In the present study, oxygen-glucose deprivation and subsequent reoxygenation (OGD/R) in H9c2 heart-derived myocytes was used as a model to study myocardial IR injury in vitro. mRNA and protein expression levels were determined by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The levels of reactive oxygen species were measured using the fluorescence dye 2,7-dichlorodihydrofluorescein diacetate. The present data demonstrated that p21 expression was upregulated by tumor protein p53 (p53) in H9c2 cells exposed to OGD/R. p21 protected H9c2 cells against OGD/R-induced oxidative stress. In addition, p21 mediated upregulation of NF-E2-related factor-2 (Nrf2), a regulator of antioxidant responses, which in turn suppressed cell death in H9c2 cells subjected to OGD/R. Thus, activation of the p53/p21/Nrf2 signaling pathway may be an important adaptive response that limits oxidative injury during IR.