The phospholipase A₂ (PLA₂) superfamily hydrolyzes phospholipids to release free fatty acids and lysophospholipids, some of which can mediate inflammation and demyelination, hallmarks of the CNS autoimmune disease multiple sclerosis. The expression of two of the intracellular PLA₂s (cPLA₂ GIVA and iPLA₂ GVIA) and two of the secreted PLA₂s (sPLA₂ GIIA and sPLA₂ GV) are increased in different stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We show using small molecule inhibitors, that cPLA₂ GIVA plays a role in the onset, and iPLA₂ GVIA in the onset and progression of EAE. We also show a potential role for sPLA₂ in the later remission phase. These studies demonstrate that selective inhibition of iPLA₂ can ameliorate disease progression when treatment is started before or after the onset of symptoms. The effects of these inhibitors on lesion burden, chemokine and cytokine expression as well as on the lipid profile provide insights into their potential modes of action. iPLA₂ is also expressed by macrophages and other immune cells in multiple sclerosis lesions. Our results therefore suggest that iPLA₂ might be an excellent target to block for the treatment of CNS autoimmune diseases, such as multiple sclerosis.