Aim: To examine the neuroprotective effects of the Toll-like receptor 3 (TLR3) agonist Poly I:C in acute ischemic models in vitro and in vivo. Methods: Primary astrocyte cultures subjected to oxygen-glucose deprivation (OGD) were used as an in vitro simulated ischemic model. Poly I:C was administrated 2 h before OGD. Cell toxicity was measured using MTT assay and LDH leakage assay. The levels of TNF alpha, IL-6 and interferon-beta (IFN beta) in the media were measured using ELISA. Toll/interleukin receptor domain-containing adaptor-inducing IFN beta (TRIF) protein levels were detected using Western blot analysis. A mouse middle cerebral artery occlusion (MCAO) model was u sed for in vivo study. The animals were administered Poly I:C (0.3 mg/kg, im) 2 h before MCAO, and examined with neurological deficit scoring and TTC staining. The levels of TNF alpha and IL-6 in ischemic brain were measured using ELISA. Results: Pretreatment with Poly I:C (10 and 20 mu g/mL) markedly attenuated OGD-induced astrocyte injury, and significantly raised the cell viability and reduced the LDH leakage. Poly I:C significantly upregulated TRIF expression accompanied by increased downstream IFN beta production. Moreover, Poly I:C significantly suppressed the pro-inflammatory cytokines TNF alpha and IL-6 production. In mice subjected to MCAO, administration of Poly I:C significantly attenuated the neurological deficits, reduced infarction volume, and suppressed the increased levels of TNF alpha and IL-6 in the ischemic striatum and cortex. Conclusion: Poly I:C pretreatment exerts neuroprotective and anti-inflammatory effects in the simulated cerebral ischemia models, and the neuroprotection is at least in part due to the activation of the TLR3-TRIF pathway.