Cross-linking of proteins by mammalian transglutaminases (TGs) plays important roles in physiological phenomena such as blood coagulation and skin formation. We show that Drosophila TG suppressed innate immune signaling in the gut. RNA interference (RNAi) directed against TG reduced the life span of flies reared under conventional nonsterile conditions but not of those raised under germ-free conditions. In conventionally reared flies, TG RNAi enhanced the expression of genes encoding antimicrobial peptides in the immune deficiency (IMD) pathway. Wild-type flies that ingested gut lysates prepared from conventionally reared TG RNAi-treated flies had shorter life spans. In conventionally reared flies, TG RNAi triggered apoptosis in the gut and induced the nuclear translocation of Relish, the NF-κB (nuclear factor κB)-like transcription factor of the IMD pathway. Wild-type flies that ingested synthetic amine donors, which inhibit the TG-catalyzed protein-protein cross-linking reaction, showed nuclear translocation of Relish and enhanced expression of genes encoding IMD-controlled antimicrobial peptide genes in the gut. We conclude that TG-catalyzed Relish cross-linking suppressed the IMD signaling pathway to enable immune tolerance against commensal microbes.