Toll like receptor (TLR) signalling is central in controlling innate immune responses, with dysregulation of TLR pathways associated in both autoimmune and inflammatory diseases. Numerous reports suggest that cannabinoids, known to exhibit general immunosuppressive properties, have potential therapeutic value in the treatment of inflammatory conditions. In relation to multiple sclerosis (M.S.), cannabinoids palliate M.S. patient symptoms and the severity of clinical signs associated with experimental autoimmune encephalomyelitis (EAE), an animal model of M.S. However, the precise molecular mechanism for these effects is not understood. While the synthetic cannabinoid R(+)WIN55,212-2 exerts established anti-inflammatory effects in vitro and in vivo, the role of cannabinoid compounds in modulating TLR signalling events is unknown. Here, using a variety of cell model systems, evidence is provided that R(+)WIN55,212-2, in the presence of the TLR3 ligand, Poly(I:C), targets interferon regulatory factor 3 (IRF3), a transcription factor essential for regulating type I interferon (IFN) expression. These findings also demonstrate that protective effects of R(+)WIN55,212-2 in EAE mice exist, as evidenced by reduced clinical scores, demyelination and inflammation. Furthermore, the anti-inflammatory effects of R(+)WIN55,212-2 in this model are IFN- dependent. In addition to blunting pro-inflammatory signaling induced by Poly(I:C) in PBMCs from both healthy donors and M.S. patients, R(+)WIN55,212-2 robustly enhanced IFN- production in these cells, an event restricted to M.S. patients. This study also highlights the anti-inflammatory potential of R(+)WIN55,212-2 by virtue of its inhibitory effects on the NFB pathway. R(+)WIN55,212-2 can inhibit TLR3/4-induced activation of NFB. This likely makes a major contribution to the inhibitory effects of R(+)WIN55,212-2 on pro-inflammatory gene expression. Indeed, it is also demonstrated that R(+)WIN55,212-2 blunts TLR3/4 induction of TNF-. The regulatory effects of R(+)WIN55,212-2 on TLR3/TLR4-induced activation of IRF3, IFN- and NFκB are independent of the cannabinoid receptors and evidenced is presented suggesting a potential role for peroxisome proliferator activated receptors in mediating the regulatory effects R(+)WIN55,212-2. Overall these findings highlight that R(+)WIN55,212-2, by targeting IRF3 and IFN- downstream of TLR3 stimulation, may exert anti-inflammatory properties and provide evidence that cannabinoid administration may offer therapeutic potential in the treatment of M.S.