Aerobic interval training (AIT) exerts beneficial effects on cardiovascular disease. However, its cardioprotective mechanisms are not fully understood. This study was designed to evaluate AIT‐mediated antioxidation by focusing on antioxidase and mitochondrial biogenesis in rats post‐myocardial infarction (MI). Sprague‐Dawley rats were divided into three groups: sham‐operated (CON), MI and MI+AIT. Myocardial microstructure and function, oxidative stress markers, mitochondrial antioxidase, phase II enzymes and mitochondrial biogenesis were assessed. The nuclear factor‐erythroid 2‐related factor (Nrf) and phosphorylated 5’‐AMP‐activated kinase (AMPK) levels were determined. The antioxidative gene sirtuin (SIRT) 3 and pro‐survival phosphatidylinositol‐3 kinase (PI3K)‐protein kinases B (Akt) signaling cascades were also evaluated. Compared with CON, post‐MI rats demonstrated noticeable microstructure injury, cardiac dysfunction and oxidative damage. In addition, decreased mitochondrial antioxidase contents, phase II enzyme (except heme oxygenase‐1) expressions and mitochondrial biogenesis were observed as well as reduced protein levels of the regulators Nrf2 and phosphorylated AMPK. SIRT3 levels and PI3K‐Akt signaling were also suppressed. These detrimental modifications were considerably ameliorated by AIT, as evidenced by increased antioxidase and elevated mitochondrial biogenesis. Nrf2 and AMPK phosphorylation were also increased after AIT, accompanied by SIRT3 upregulation and PI3K‐Akt signaling activation. Moreover, PI3K inhibitor treatment partly attenuated AIT‐elicited increased Nrf2 levels and AMPK phosphorylation.AIT can effectively alleviate MI‐induced oxidative injury, which may closely correlate with antioxidase system activation and mitochondrial biosynthesis. Increased SIRT3 expression and PI3K‐Akt signaling activation may play key roles in AIT‐mediated antioxidation. These results open up new avenues for exercise intervention therapies for MI patients. This article is protected by copyright. All rights reserved.