Th1 cells are critical in experimental autoimmune encephalomyelitis (EAE). Serpine1 has been posited as an inhibitor of IFN? from T cells though its role in autoimmunity remains unclear. Here, we show thatSerpine1-/-mice develop EAE of enhanced severity relative to wild-type controls.In vitro, Serpine1 transcript and protein are upregulated in Th1 cells.In vivo,Serpine1-/-x 2D2 Th1 cells transfer EAE of increased severity in comparison toSerpine1+/+x 2D2 Th1 cells. Notably, polarizedSerpine1-/-Th1 cells display delayed expression of the Th1-specific inhibitory receptor, Tim-3.Serpine1-/-Tim-3-Tg Th1 cells, which transgenically over-express Tim-3, showed increased expression of IFN? and reduced expression of the checkpoint molecules Lag-3 and PD-1. Further, Serpine1 deficiency restored the EAE phenotype of Tim-3-Tg mice that normally develop mild disease. Together, we identify Serpine1 as a negative regulator of Th1 cells.Key pointsSerpine1 inhibits EAE in a T cell-dependent manner.Serpine1 is upregulated in Th1 cells and inhibits their pathogenicity.Serpine1 promotes expression and function of Th1-specific inhibitory receptor Tim-3.