Ischemia/reperfusion (I/R) commonly causes dysfunction of the blood-brain barrier (BBB). Restoration of the BBB function can relieve neurologic damage caused by I/R brain injuries. Hypothermia is one of the most potent therapeutics against ischemic brain injury. However, mechanisms of BBB protection in therapeutic hypothermia are still unclear. Recent reports have suggested that SUMO-conjugation of proteins may provide neuroprotection against ischemic brain injury. We therefore evaluated the effects of SUMOylation on the BBB permeability in therapeutic hypothermia for ischemic brain injury. Syrian hamsters were subjected to 30 min of transient bilateral carotid common artery occlusion (tBCCAO). BBB permeability was assessed using Evans blue (EB) extravasation method. Therapeutic hypothermia reduced EB extravasation caused by tBCCAO treatment and induced a marked increase in levels of SUMO2/3-conjugated proteins. BBB permeability protected by hypothermia was impaired by the pretreatment of anacardic acid, an inhibitor of SUMO-activating enzyme E1. Furthermore, the pretreatment of anacardic acid increased the expression levels of matrix metalloproteinase-9. These findings suggest that therapeutic hypothermia protects against ischemic brain injury by maintenance of BBB function via increase in SUMOylation levels.