Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. Toll-like receptor (TLR)-mediated signalling plays a role in cerebral ischaemia/reperfusion (I/R) injury. Modulation of TLRs has been reported to protect against cerebral I/R injury. This study examined whether modulation of TLR3 with poly (I:C) will induce protection against cerebral I/R injury. Mice were treated with or without Poly (I:C) (n = 8/group) 1 hr prior to cerebral ischaemia (60 min.) followed by reperfusion (24 hrs). Poly (I:C) pre-treatment significantly reduced the infarct volume by 57.2% compared with untreated I/R mice. Therapeutic administration of Poly (I:C), administered 30 min. after cerebral ischaemia, markedly decreased infarct volume by 34.9%. However, Poly (I:C)-induced protection was lost in TLR3 knockout mice. In poly (I:C)-treated mice, there was less neuronal damage in the hippocampus compared with untreated I/R mice. Poly (I:C) treatment induced IRF3 phosphorylation, but it inhibited NF-κB activation in the brain. Poly (I:C) also decreased I/R-induced apoptosis by attenuation of Fas/FasL-mediated apoptotic signalling. In addition, Poly (I:C) treatment decreased microglial cell caspase-3 activity. In vitro data showed that Poly (I:C) prevented hypoxia/reoxygenation (H/R)-induced interaction between Fas and FADD as well as caspase-3 and -8 activation in microglial cells. Importantly, Poly (I:C) treatment induced co-association between TLR3 and Fas. Our data suggest that Poly (I:C) decreases in cerebral I/R injury via TLR3 which associates with Fas, thereby preventing the interaction of Fas and FADD, as well as microglial cell caspase-3 and -8 activities. We conclude that TLR3 modulation by Poly (I:C) could be a potential approach for protection against ischaemic stroke.