Previously we reported that IL-17-producing CD4 T cells (Th17) were increased in mice lacking the protease inhibitor SerpinB1 and several SerpinB1-inhibitable cysteine cathepsins were induced in the Th17 cells, most prominently cathepsin L (CtsL). Since CtsL also mediates invariant chain processing in thymic epithelial cells, deficiency of CtsL leads to impaired CD4 T cell thymic selection, which hinders the direct investigation of CD4 T cells in CtsL -/- mouse. In the current study, through transplanting the CtsL -/- bone marrow into lethally irradiated CtsL-sufficient Rag/- mice (bone marrow chimeras), we reconstituted the immune system of CtsL -/- chimeric mice, which possessed normal CD4 T cell development and allowed us to study the intrinsic role of CtsL in CD4 T cells in Th17 cell-driven autoimmune diseases. Surprisingly, we found that CtsL -/- CD4 T cells had no defects in differentiation of naïve CD4 T cells into Th1, Treg and Th17 cells in vitro. However, in vivo, in experimental autoimmune encephalomyelitis (EAE) model, deficiency of CtsL significantly decreased the activation of IL-17, GM-CSF and IFN-γ producing pathogenic CD4 T cells. Compared with wild type (wt) controls, CtsL -/- CD4 T cells were also less accumulated in the spinal cord in EAE. Thus, for the first time, our study provided the direct in vivo evidence that CtsL was involved in CD4 T cells acquiring pathogenicity in the autoimmune disease.