Background: Cerebral ischemia reperfusion (CIR) affects microRNA (miR) expression and causes substantial inflammation. Here, we investigated the influence and underlying mechanism of miR-27-3p in rats with CIR.Methods: Cerebral ischemia reperfusion was built by tMCAO. Rats were randomly divided into Sham group, brain ischemic reperfusion (IR) group, brain ischemic reperfusion transfected with NC group, brain ischemic reperfusion transfected with miR-27a-3p mimic group, brain ischemic reperfusion transfected with miR-27a-3p inhibitor group and brain ischemic reperfusion transfected with miR-27a-3p mimic and Litaf mimic group. The relationship between miR-27a-3p and Litaf was verified via qRT-PCR and luciferase assays. The cellular distribution of Litaf was determined via double immunofluorescence. The effect of miR-27a-3p on the expression of Litaf, TLR4, NF-κB and IL-6 was evaluated using synthetic miR-27a-3p mimic and inhibitor. The level of Nissl’s body in each group was detected by Nissl’s staining. The infarct in each group was evaluated by TTC staining.Results: Firstly, BV treatment relieves cerebral infarction and decreases the levels of serum IL-1β, IL-6 and TNF-α. Through our previous study, we found key microRNA mR-27a-3p and its targeted gene Litaf might involve in the molecular mechanism of CIR. Then, the regulation between miR-27a-3p and Litaf was verified by the temporal miR-27a-3p and Litaf expression profiles and luciferase assay. Moreover, intracerebroventricular injection of the miR-27a-3p mimic significantly decreased the Litaf, TLR4, NF-κB and interleukin (IL)-6 levels and the double-labeled cell count 24 h post-surgery, whereas miR-27a-3p inhibitor reversed these effects. Furthermore, miR-27a-3p mimic could relive cerebral infarct and neurologic deficit after CIR. In addition, injection of miR-27a-3p mimic decreased neuronal damage induced by CIR.Conclusions: Increasing miR-27-3p levels protect against CIR by relieving inflammation, neuronal damage and neurologic deficit by inhibiting LITAF and the TLR4/NF-κB pathway.