The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies. Synopsis The protease activity of MALT1 is essential for the adaptive immune response, but also for the generation of Treg cells and the prevention of autoimmune gastritis. Mice expressing a catalytically inactive form of Malt1 (Malt1 knock-in mice) are strongly immunodeficient and have impaired development of marginal zone B cells and B1 B cells. Malt1 protease activity is required for efficient activation of lymphocytes, NK cells, and dendritic cells by immunoreceptors with ITAM motifs. The absence of Malt1 protease activity protects mice against experimental autoimmune encephalitis and T-cell transfer-induced colitis. The protease activity of Malt1 is also essential for the development of natural regulatory T cells (Tregs). Malt1 knock-in mice but not Malt1-deficient mice develop autoimmune gastritis, most likely as a consequence of Malt1 scaffold-driven immune responses in the absence of efficient Treg functions. The protease activity of MALT1 is essential for the adaptive immune response, the generation of Treg cells, and the prevention of autoimmune gastritis.