We have demonstrated that R(+)WIN55,212-2, a synthetic cannabinoid that possesses cannabimimetic properties, acts as a novel regulator of Toll-like receptor 3 (TLR3) signaling to interferon (IFN) regulatory factor 3 (IRF3) activation and IFN-Î²expression, and this is critical for manifesting its protective effects in a murine multiple sclerosis model. Here we investigated the role of peroxisome proliferator-activated receptor-Î±(PPARÎ±) in mediating the effects of R(+)WIN55,212-2 on this pathway. Data herein demonstrate that the TLR3 agonist poly(I:C) promotes IFN-Î² expression and R(+)WIN55,212-2 enhances TLR3-induced IFN-Î² expression in a stereoselective manner via PPARÎ±. R(+)WIN55,212-2 promotes increased transactivation and expression of PPARÎ±. Using the PPARÎ±antagonist GW6471, we demonstrate that R(+)WIN55,212-2 acts via PPARÎ± to activate JNK, activator protein-1, and positive regulatory domain IV to transcriptionally regulate the IFN-Î²promoter. Furthermore, GW6471 ameliorated the protective effects of R(+)WIN55,212-2 during the initial phase of experimental autoimmune encephalomyelitis. Overall, these findings define PPARÎ± as an important mediator in manifesting the effects of R(+)WIN55,212-2 on the signaling cascade regulating IFN-Î² expression. The study adds to our molecular appreciation of potential therapeutic effects of R(+)WIN55,212-2 in multiple sclerosis. Â© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.