Update publication information. Previous studies indicated that the ras-related C3 botulinum toxin substrate 1 (RAC1), a member of the Rac family of guanosine triphosphate phosphohydrolases (GTPase), is an important regulator of myocardial ischemia and reperfusion (I/R) injury. Potential interaction between the signal-induced proliferation-associated 1 (SPA-1) and RAC1 has been observed in cancer and neurological diseases, but the role of SPA-1 in myocardial I/R injury has not been fully investigated. Using iTRAQ-based proteomic analysis, we found that SPA-1 protein expression was moderately upregulated and RAC1 was significantly activated in H9C2 cells treated with hypoxia and reoxygenation (H/R). Moreover, others have shown that inhibiting SPA-1 with siRNA and overexpressing SPA-1 with plasmid transfection moderately upregulated SPA-1 during H/R which may be protect against H/R related cardiomyocyte injury and subsequent cytoskeleton malformation, cardiac fibrosis, apoptosis, and cellular reactive oxygen species formation. Finally, in a rat model of myocardial I/R induced by left anterior descending artery ligation, adenovirus-mediated overexpression of SPA-1 significantly alleviated the pathohistological changes in the myocardium and inhibited RAC1 activation. Taken together, these results indicate that under I/R conditions, moderate upregulation of SPA-1 may be a self-regulated cardioprotective response against RAC1-mediated injuries via attenuating multiple pathophysiological processes. Targeted enhancing of SPA-1 protein expression and suppressing RAC1 activation may be an important treatment strategy for preventing myocardial I/R injury.