Ischemia/reperfusion (I/R) is a common injury leading to ischemic stroke. At present, I/R treatment remains limited, highlighting the urgent need for the discovery and development of new protective drugs for brain injury. Here, we investigated the neuroprotective effects of short peptide OM-LV20 previously identified from amphibian against I/R rats. Results showed that intraperitoneal administration of OM-LV20 (20 ng/kg) significantly reduced infarct area formation, improved behavioral abnormalities, and protected cortical and hippocampal neurons against death caused by I/R. Moreover, the underlying molecular mechanism was involved with the regulation of the MAPK and BDNF/AKT signaling pathways, as well as the levels of cyclic adenosine monophosphate, pituitary adenylate cyclase-activating polypeptide receptor, and tryptophan hydroxylase 1. To the best of our knowledge, this research was the first report to describe the neuroprotective effects of an amphibian skin secretion-derived peptide in I/R rats and highlighted OM-LV20 as a promising drug candidate for the development of novel anti-stroke therapies. © 2020 Elsevier Inc.