Mild hypothermia has a protective effect on ischemic stroke, but the mechanisms remain elusive. Here, we investigated microRNA (miRNA) profiles and the specific role of miRNAs in ischemic stroke treated with mild hypothermia. Male adult Sprague Dawley rats were subjected to focal transient cerebral ischemia. Mild hypothermia was induced by applying ice packs around the neck and head of the animals. miRNAs expression profiles were detected in ischemic stroke treated with mild therapeutic hypothermia through miRNA chips. Reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the change of miRNA array. Western blot and adenosine triphosphate (ATP) assay kits were used to detect the changes of protein expression and ATP levels, respectively. miR-15b mimic and its control were injected into the right lateral ventricle 60 min before the induction of ischemia. The results showed that mild hypothermia affected miRNAs profiles expression. We verified the expression of miR-15b and miR-598-3p by miRNA RT-PCR. miR-15b mimic inhibited the expression of its target, ADP ribosylation factor-like 2 (Arl2) protein, and decreased ATP levels in PC12 cells. Compared with the control, miR-15b mimic increased the infarct volume and aggravated the neurological function under normothermia or hypothermia treatment. Furthermore, the expression of Arl2 was decreased in the miR-15b mimic group under normothermia or hypothermia treatment. Mild therapeutic hypothermia affected miRNA profiles and protected against cerebral ischemia/reperfusion by inhibiting miR-15b expression in rats. miR-15b may be a potential target for therapeutic intervention in stroke.