DNA2, a multifunctional enzyme with structure-specific nuclease, 5 -to-3 helicase, and DNA-dependent ATPase activities, plays a pivotal role in the cellular response to DNA damage. However, its involvement in cerebral ischemia/reperfusion (I/R) injury remains to be elucidated. This study investigated the involvement of DNA2 in cerebral I/R injury using conditional knockout (cKO) mice ( -Cre) subjected to middle cerebral artery occlusion (MCAO), an established model of cerebral I/R. Results demonstrated a gradual up-regulation of DNA2 expression, peaking at 72 h post-MCAO. Notably, cKO mice exhibited more pronounced brain injury, neurological deficits, and neuronal apoptosis within the penumbra following MCAO. Additionally, DNA2 expression was elevated in an oxygen-glucose deprivation/reoxygenation (OGD/R) cell culture model, and knockdown (KD) exacerbated neuronal apoptosis and oxidative stress. Transcriptome analysis of ischemic penumbra tissues via RNA sequencing revealed significant down-regulation of in cKO mice. Furthermore, experiments demonstrated that overexpression of ameliorated KD-induced neuronal apoptosis. Collectively, these findings demonstrate that deficiency exacerbates cerebral I/R injury through the down-regulation of , highlighting a novel regulatory axis in ischemic neuroprotection.