This study reveals that S-palmitoylation regulates TLR9 signaling and systemic autoimmunity in mice. Palmitoyl-protein thioesterase 1 (PPT1) removes S-palmitoylation from TLR9 in lysosomes, thereby modulating TLR9 response and autoimmunity. PPT1 promotes IFNα secretion by plasmacytoid dendritic cells (pDCs) and TNF secretion by macrophages. Genetic deficiency or chemical inhibition of PPT1 reduces anti-nuclear antibody levels and attenuates nephritis in B6.Sle1yaa mice. In human SLE patients, the PPT1 inhibitor HDSF reduces IFNα production ex vivo. Mechanistically, TLR9 is S-palmitoylated at C258 and C265, and the protein acyltransferase DHHC3 palmitoylates TLR9 in the Golgi, regulating its trafficking to endosomes. Subsequent depalmitoylation by PPT1 facilitates the release of TLR9 from UNC93B1. The study identifies a posttranslational modification cycle that controls TLR9 response and autoimmunity. TLR9 and TLR7 are S-palmitoylated, with DHHC3 palmitoylating TLR9 in the Golgi and PPT1 depalmitoylating it in lysosomes. PPT1 deficiency or inhibition reduces autoantibody levels and attenuates SLE pathogenesis. The palmitoylation cycle regulates TLR9 trafficking and depalmitoylation, which controls TLR9 release from UNC93B1. The study highlights the importance of palmitoylation in TLR9 signaling and suggests PPT1 as a potential therapeutic target for autoimmune diseases.This study reveals that S-palmitoylation regulates TLR9 signaling and systemic autoimmunity in mice. Palmitoyl-protein thioesterase 1 (PPT1) removes S-palmitoylation from TLR9 in lysosomes, thereby modulating TLR9 response and autoimmunity. PPT1 promotes IFNα secretion by plasmacytoid dendritic cells (pDCs) and TNF secretion by macrophages. Genetic deficiency or chemical inhibition of PPT1 reduces anti-nuclear antibody levels and attenuates nephritis in B6.Sle1yaa mice. In human SLE patients, the PPT1 inhibitor HDSF reduces IFNα production ex vivo. Mechanistically, TLR9 is S-palmitoylated at C258 and C265, and the protein acyltransferase DHHC3 palmitoylates TLR9 in the Golgi, regulating its trafficking to endosomes. Subsequent depalmitoylation by PPT1 facilitates the release of TLR9 from UNC93B1. The study identifies a posttranslational modification cycle that controls TLR9 response and autoimmunity. TLR9 and TLR7 are S-palmitoylated, with DHHC3 palmitoylating TLR9 in the Golgi and PPT1 depalmitoylating it in lysosomes. PPT1 deficiency or inhibition reduces autoantibody levels and attenuates SLE pathogenesis. The palmitoylation cycle regulates TLR9 trafficking and depalmitoylation, which controls TLR9 release from UNC93B1. The study highlights the importance of palmitoylation in TLR9 signaling and suggests PPT1 as a potential therapeutic target for autoimmune diseases.