Background and Purpose Myocardial ischemia/reperfusion (MI/R) injury is the main clinical problem of coronary heart disease. Higenamine (HG), a cardiotonic active component isolated from aconiti tuber, exhibits cardioprotective effects. However, its exact mechanism in MI/R is limited. Experimental Approach The oxidative stress induced by H2O2 in H9c2 and MI/R rat model was established. Metprolol, evidenced to ameliorate MI/R injury, was used as the positive control. Cardiac impairment indexes, were detected in vitro and in vivo. Changes of peroxiredoxins (Prxs), important anti-oxidative stress proteins in MI/R were also evaluated. Adenovirus induced Prx2 down-regulation and specific inhibitor of Prx2 was employed to determine the exact mechanism of HG in vitro and in vivo. Metabolomic analysis of MI/R rats with or without HG administration based on UPLC-MS/MS was further performed. Key Results HG significantly reduced the oxidative stress, improve cardiac dysfunction and decrease cardiac infarct size against MI/R via Prx2 activation, which was stronger than those in positive control treated rats. Additionally, HG altered metabolism of arginine and proline and biosynthesis of arginine during MI/R. Mechanically, HG increased the levels of Prx2, accompanied with decreased apoptotic protein, in vitro and in vivo expressions. However, down-regulation of Prx2 markedly blocked all the cardioprotective effect of HG. Conclusion and Implications Our work systematicly demonstrated that HG possessed a strong cardioprotective effect against MI/R by up-regulating cardiac expression of Prx2 and modulating disordered metabolism of arginine and glutamine in heart. We provided HG as the potential to further explore therapeutic strategy for MI/R.