The role played by apoptosis in the homeostasis of effector cells of the innate immune system is unclear. Serine protease inhibitor 6 (Spi6) is an inhibitor of granzyme B (GrB) that protects cytotoxic T lymphocytes of the adaptive immune system from apoptosis. To determine whether Spi6 also protects cells of the innate immune system from self-inflicted damage we have examined invariant NKT (iNKT) cells. Spi6-deficient iNKT cells harbored increased levels of GrB after TCR stimulation with the PBS-57 glycolipid Ag and were susceptible to apoptosis. The increased apoptosis of Spi6 knock-out (KO) iNKT cells lead to a complete loss in the production of IL-4 and IFN-gamma by Spi6 KO iNKT cells after PBS-57 challenge. The increased activation-induced apoptosis resulted in impaired survival and a decreased clonal burst size of Spi6 KO iNKT cells, which could be corrected by GrB deficiency. However, the clonal burst of Spi6 KO iNKT cells after TCR-independent activation with lymphocytic choriomeningitis virus was not affected. Our findings demonstrate that Spi6 protects cytotoxic cells of the innate immune system from GrB-mediated self-inflicted triggered by the recognition of Ag.