Intestinal ischemia/reperfusion injury (IIRI) has the potential to be life threatening and is associated with significant morbidity and serious damage to distant sites in the body on account of disruption of the intestinal mucosal barrier. In the present study, we have explored this line of research by comparing and identifying peptides that originated from the intestinal segments of IIRI model rats by using liquid chromatography-mass spectrometry (LC-MS). We also analyzed the basic characteristics, cleavage patterns, and functional domains of differentially expressed peptides (DEPs) between the IIRI model rats and control (sham-operated) rats and identified bioactive peptides that are potentially associated with ischemia reperfusion injury. We also performed bioinformatics analyses in order to identify the biological roles of the DEPs based on their precursor proteins. Enrichment analysis demonstrated the role of several DEPs in impairment of the intestinal mucosal barrier caused by IIRI. Based on the results of comprehensive ingenuity pathway analysis, we identified the DEPs that were significantly correlated with IIRI. We identified a candidate precursor protein (Actg2) and seven of its peptides, and we found that Actg2-6 had a more significant difference in its expression, a longer half-life, and better lipophilicity, hydrophobicity, and stability than the other candidate Actg2 peptides examined. Furthermore, we observed that Actg2-6 might play critical roles in the protection of the intestinal mucosal barrier during IIRI. In summary, our study provides a better understanding of the peptidomics profile of IIRI, and the results indicate that Actg2-6 could be a useful target in the treatment of IIRI.