Background Despite considerable efforts, ischemic stroke (IS) remains a challenging clinical problem. Therefore, the discovery of effective therapeutic and targeted drugs based on the underlying molecular mechanism is crucial for effective IS treatment. Methods A cDNA-encoding peptide was cloned from RNA extracted from Rana limnocharis skin, and the mature amino acid sequence was predicted and synthesized. Hemolysis and acute toxicity of the peptide were tested. Furthermore, its neuroprotective properties were evaluated using a middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats and an oxygen-glucose deprivation/reperfusion (OGD/R) model in neuron-like PC12 cells. The underlying molecular mechanisms were explored using microRNA (miRNA) sequencing, quantitative real-time polymerase chain reaction, dual-luciferase reporter gene assay, and western blotting. Results A new peptide (NP1) with an amino acid sequence of 'FLPAAICLVIKTC' was identified. NP1 showed no obvious toxicities in vivo and in vitro and was able to cross the blood-brain barrier. Intraperitoneal administration of NP1 (10 nmol/kg) effectively reduced the volume of cerebral infarction and relieved neurological dysfunction in MCAO/R model rats. Moreover, NP1 significantly alleviated the decrease in viability and increase in apoptosis of neuron-like PC12 cells induced by OGD/R. NP1 effectively suppressed inflammation by reducing interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) levels in vitro and in vivo. Furthermore, NP1 up-regulated the expression of miR-6328, which, in turn, down-regulated kappa B kinase beta (IKK beta). IKK beta reduced the phosphorylation of nuclear factor-kappa B p65 (NF-kappa B p65) and inhibitor of NF-kappa B (I-kappa B), thereby inhibiting activation of the NF-kappa B pathway. Conclusions The newly discovered non-toxic peptide NP1 ('FLPAAICLVIKTC') exerted neuroprotective effects on cerebral ischemia-reperfusion injury by reducing inflammation via the miR-6328/IKK beta/NF-kappa B axis. Our findings not only provide an exogenous peptide drug candidate and endogenous small nucleic acid drug candidate but also a new drug target for the treatment of IS. This study highlights the importance of peptides in the development of new drugs, elucidation of pathological mechanisms, and discovery of new drug targets.