The proteinase-activated receptors (PARs) are widely recognized for their modulatory properties of inflammation and neurodegeneration. We investigated the role of PAR2 in the pathogenesis of multiple sclerosis (MS) in humans and experimental autoimmune encephalomyelitis (EAE) in mice. PAR2 expression was increased on astrocytes and infiltrating macrophages in human MS and murine EAE central nervous system (CNS) white matter (P < 0.05). Macrophages and astrocytes from PAR2 wild-type (WT) and knockout (KO) mice exhibited differential immune gene expression with PAR2 KO macrophages showing significantly higher interleukin 10 production after lipopolysaccharide stimulation (P < 0.001). PAR2 activation in macrophages resulted in the release of soluble oligodendrocyte cytotoxins (P < 0.01). Myelin oligodendrocyte glycoprotein-induced EAE caused more severe inflammatory gene expression in the CNS of PAR2 WT animals (P < 0.05), together with enhanced T cell proliferation and interferon gamma production (P < 0.05), compared with KO littermates. Indeed, PAR2 WT animals showed markedly greater microglial activation and T lymphocyte infiltration accompanied by worsened demyelination and axonal injury in the CNS compared with their PAR2 KO littermates. Enhanced neuropathological changes were associated with a more severe progressive relapsing disease phenotype (P < 0.001) in WT animals. These findings reveal previously unreported pathogenic interactions between CNS PAR2 expression and neuroinflammation with ensuing demyelination and axonal injury.