Objectives: The protective effects of hypothermia on acute stroke have been demonstrated in many studies. However, its underlying mechanisms have not been thoroughly elucidated. Following an ischemic stroke event, microglia undertakes an early 'healthy' M2 phenotype and gradually transform into a 'sick' M1 phenotype over time. This transformation of polarity of microglia has influence on the degree of damage following a stroke. This study investigated the effects of mild focal hypothermia on microglia polarization following ischemic stroke. Methods: Transient cerebral ischemic models were created by intraluminal filament occlusion of right middle cerebral artery (MCAO) in mice for one hour. By placing an ice box under their skull, hypothermia of mice brain was initiated immediately following MCAO for 2 h. Temporal muscle temperature was recorded and maintained between 32 and 34 degrees C. Brain tissue loss was assessed by hematoxylin and eosin (H&E) staining 28 days after MCAO. Quantitative real-time polymerase chain reaction (qPCR) and immunostaining were used to assess phenotype of microglia in different ischemic perfusion time. Results: Hypothermia reduced brain tissue loss 28 days after ischemic stroke. Hypothermia also reduced the number of CD16-positive M1 microglia and increased the numbers of CD206-positive M2 microglia following ischemic stroke. Moreover, hypothermia also led to the reduction of the M1 markers at the level of transcription, while it increased the expression of mRNA for M2 markers. Conclusions: Hypothermia is protective following ischemic stroke and can reduce brain tissue loss. Moreover, hypothermia shifts the polarization of microglia from the M1 to the M2 phenotype in the ischemic mice brain. This observed biological phenomenon may partially explain the protective effects seen due to hypothermia in acute ischemic stroke.