How hypothermia serves an early protective role against cerebral ischemia remains to be determined. The small ubiquitin-related modifier protein (SUMO) functions as a post-translational modification system and SUMO-2/3 subtypes are often activated in early stress. The present study investigated changes in the protein level of SUMO using western blotting and immunocytochemistry when neurons were exposed to oxygen-glucose deprivation (OGD) in vitro, as well as in a rat model of middle cerebral artery occlusion (MCAO) in vivo. The results demonstrated that a large number of proteins were conjugated to SUMO-2/3 in OGD-injured neurons (within 10 min, peaking at 12 h), and was markedly enhanced under conditions of hypothermia (33 degrees C). Concordantly, lactate dehydrogenase (LDH) release and the apoptosis rate, as determined by LDH and TUNEL assays, respectively, were lower in hypothermia-treated neurons. Similar results were obtained in a rat model of MCAO. Neurological deficit scores were lower in the hypothermia group than in the sham group in the early stage of cerebral ischemia (P< 0.05). However, no significant differences in neurological deficit scores were detected between the hypothermia group and the sham group in the late stage of ischemia (21 days; P> 0.05). This study implicates a role for SUMO-2/3 in early hypothermia-induced neuroprotection against stroke. The development of small molecule therapeutics based on SUMO-2/3 may benefit patients with cerebral ischemia.