The class IB phosphoinositide 3-kinase gamma enzyme complex (PI3K) functions in multiple signaling pathways involved in leukocyte activation and migration, making it an attractive target in complex human inflammatory diseases including MS. Here, using pik3cg(-/-) mice and a selective PI3K inhibitor, we show that PI3K promotes development of experimental autoimmune encephalomyelitis (EAE). In pik3cg(-/-) mice, EAE is markedly suppressed and fewer leukocytes including CD4(+) and CD8(+) T cells, granulocytes and mononuclear phagocytes infiltrate the CNS. CD4(+) T cell priming in secondary lymphoid organs is reduced in pik3cg(-/-) mice following immunisation. This is attributable to defects in DC migration concomitant with a failure of full T cell activation following TCR ligation in the absence of p110. Together, this results in suppressed autoreactive T cell responses in pik3cg(-/-) mice, with more CD4(+) T cells undergoing apoptosis and fewer cytokine-producing Th1 and Th17 cells in lymphoid organs and the CNS. When administered from onset of EAE, the orally active PI3K inhibitor AS605240 caused inhibition and reversal of clinical disease, and demyelination and cellular pathology in the CNS was reduced. These results strongly suggest that inhibitors of PI3K may be useful therapeutics for MS.