Ischemic stroke is a devastating CNS insult with few clinical cures. Poor understanding of underlying mechanistic network is the primary limitation to develop novel curative therapies. Extracellular accumulation of granzyme B subsequent ischemia promotes neurodegeneration. Inhibition of granzyme B can be one of the potent strategies to mitigate neuronal damage. In present study, we investigated the effect of murine Serpina3n and human (homolog) SERPINA3 against cerebral ischemia through granzyme B inactivation. Recombinant Serpina3n/SERPINA3 were expressed by transfected 293 T cells, and eluted proteins were examined for postischemic influence both in vitro and in vivo. During in vitro test, Serpina3n was found effective enough to inhibit granzyme B, while SERPINA3 was ineffectual to counter cytotoxic protease. Treatment of hypoxic culture with recombinant Serpina3n/SERPINA3 significantly increased cell viability in dosage-dependent manner, recorded maximum at the highest concentration (4 mM). Infarct volume analysis confirmed that 50 mg/kg dosage of exogenous Serpina3n was adequate to reduce disease severity, while SERPINA3 lacked behind in analeptic effect. Immunohistochemical test, western blot analysis, and protease activity assay’s results illustrated successful diffusion of applied protein to the ischemic lesion and reactivity with the target protease. Taken together, our findings demonstrate therapeutic potential of Serpina3n by interfering granzyme B-mediated neuronal death subsequent cerebral ischemia.