Objective To investigate the effects of mild hypothermia on the expression of small ubiquitin‑like modifier proteins specific protease 3 (SENP3) in mouse neuro‑blastoma (N2a) cells during oxygen‑glucose deprivation/reoxygenation (OGD/R) injury. Methods Mouse N2a cells were cultured in vitro and exposed to OGD/R to establish a model of OGD/R simulating cerebral ischemia/reperfusion and a mild hypothermia model. N2a cells were divided into three groups according to the random number table method (n=5): group sham operation (S), group OGD/R and group OGD/R + mild hypothermia (OGD/R+HT). Cell counting kit ‑8 (CCK‑8) assay was used to detect N2a cell viability after OGD/R, while the release rate of lactate dehydrogenase (LDH) was detected to evaluate N2a cytotoxicity after OGD/R. Hoechst 33258 staining was used to observe N2a cell apoptosis. Real‑time quantitative PCR (RT‑qPCR) was used to analyze the relative amounts of SENP3 mRNA. Western blot was used to measure the levels of SENP3 protein. Results Compared with group S, groups OGD/R and OGD/R+HT presented decreases in cell survival rate as well as increases in LDH release rate, the number of apoptotic cells, and the relative amounts of SENP3 mRNA and protein (P<0.05). Compared with group ODG/R, group OGD/R+HT showed increases in cell survival rate and decreases in LDH release rate, the number of apoptotic neurons, and the relative amounts of SENP3 mRNA and protein (P<0.05). Conclusions Mild hypothermia alleviates OGD/R injury in N2a cells, which is related with the inhibition of SENP3 expression.