Alpha/beta-hydrolase domain 6 (ABHD6) is a novel 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, that can fine-tune the endocannabinoid signaling in the central nervous system. Recently we and others have demonstrated the protective effect of ABHD6 inhibition in the animal models of traumatic brain injury and epileptic seizures. In this study, we investigated the role of targeting ABHD6 in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Post-symptom treatment with an ABHD6 inhibitor WWL70 increased the brain levels of 2-AG and ameliorated the clinical signs of EAE, T cells infiltration, microglia activation and the expression of activated leukocyte cell adhesion molecules. The production of iNOS, COX-2, TNF- and IL-1 and the phosphorylation of NF-B were also significantly reduced by WWL70 treatment. The neuroprotective effect of WWL70 was demonstrated by increased survival of mature oligodendrocytes, reduced demyelination and axonal loss in WWL70 treated EAE mouse spinal cord. The therapeutic effect of WWL70 on EAE was absent by co-administration of CB2 receptor antagonist, but not CB1 receptor antagonist. Consistently, WWL70 did not afford any protection in CB2 receptor knockout mice after EAE induction. Given the increased expression of ABHD6 in microglia/macrophages, but not in T cells, we speculated that inhibition of ABHD6 might enhance 2-AG signaling particularly in microglia/macrophages to exert anti-inflammatory effects via activation of CB2 receptors. These results suggest that inhibition of ABHD6 might be used as an ideal strategy for the treatment of MS and other neurodegenerative diseases.