Microglia are considered to be potential anti- gen-presenting cells and have the ability to present antigen under pathological conditions. Nevertheless, whether and how microglia are involved in immune regulation are lar- gely unknown. Here, we investigated the suppressive activity of microglia during experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendro- cyte glycoprotein, with the goal of understanding their role in regulating the T cell reaction. Using flow cytometric analysis, we found that microglia were characterized by increased cell number and up-regulated programmed death ligand-1 (PD-L1) at the peak phase of EAE. Meanwhile, both the CD4+ T cells and microglia that infiltrated the central nervous system expressed higher levels of PD1, the receptor for PD-L1, accompanied by a decline of Thl cells. In an ex vivo co-culture system, microglia from EAE mice inhibited the proliferation of antigen-specific CD4+ T cells and the differentiation of Thl cells, and this was significantly inhibited by PD-L 1 blockade. Further, microglia suppressed Thl cells via nitric oxide (NO), the production of which was dependent on PD-L1. Thus, these data suggest a scenario in which microglia are involved in the regulation of EAE by suppressing Thl-cell differenti- ation via the PD-L1-NO pathway.