SUMO-specific protease 1 (SENP1) deconjugates SUMO from modified proteins. Although post-ischemic activation of SUMO conjugation was suggested to be neuroprotective against ischemia/reperfusion (I/R) injury, the function of SENP1 in this process remained unclear. Here we show that transient middle cerebral artery occlusion in mice followed by 6, 12 and 24 h reperfusion significantly enhanced SENP1 levels in the affected brain area, independent of transcription. Consistent with the increase in SENP1, the levels of SUMO1-conjugated proteins were decreased by I/R in cortical neurons of control littermate mice, but unchanged in that of animals with conditional ablation of SENP1 gene from adult principal neurons, the SENP1flox/flox:CamKIIα-Cre (SENP1 cKO) mice. The SENP1 cKO mice exhibited a significant increase in infarct volume in the cerebral cortex and more severe motor impairment in response to I/R as compared with the control littermates. Cortical neurons from I/R-injured SENP1 cKO mice became more apoptotic than that from control littermates, as indicated by both TUNEL staining and caspase-3 activation. Overexpression of SENP1 in somatosensory cortices of adult wild-type (WT) mice suppressed I/R-induced neuronal apoptosis. We conclude that SENP1 plays a neuroprotective role in I/R injury by inhibiting apoptosis through decreasing SUMO1 conjugation. These findings reveal a novel mechanism of neuroprotection by protein desumoylation, which may help develop new therapies for mitigating brain injury associated with ischemic stroke.