The interleukin-15 (IL-15) system is important for regulating both innate and adaptive immune responses, however, its role in autoimmune disease remained unclear. Here we found that Il15-/- and Il15ra-/- mice spontaneously developed late-onset autoimmune phenotypes. CD4+ T cells of the knockout mice showed elevated autoreactivity as demonstrated by the induction of lymphocyte infiltration in the lacrimal and salivary glands when transferred into nude mice. The antigen-presenting cells in the thymic medullary regions expressed IL-15 and IL-15Rα, whose deficiency resulted in insufficient negative selection and elevated number of natural IL-17A-producing CD4+ thymocytes. These findings reveal previously unknown functions of the IL-15 system in thymocyte development, and thus a new layer of regulation in T cell-mediated autoimmunity.