Mast cells play an important role in innate immunity as well as in allergic reaction. However, regulatory mechanisms underlying mast cell-mediated innate immune responses remain largely unknown. Here we determined whether Smad3, a major signal transducer of TGF-beta, regulates innate immune response by mast cells against Gram-negative bacteria. Bone marrow-derived mast cells (BMMC) obtained from Smad3 null mutant mice showed augmented capacity to produce proinflammatory cytokines upon stimulation with a Gram-negative bacteria-associated product, LPS. In acute septic peritonitis model induced by cecal ligation and puncture, mast cell-deficient W/W(v) mice reconstituted with Smad3 null BMMC had significantly higher survival rate than W/W(v) mice reconstituted with wild-type BMMC, which was associated with higher production of proinflammatory cytokines in the peritoneal cavity. These in vitro and in vivo results suggest that Smad3 in mast cells functions as inhibitory for mast cell-mediated innate immune response against Gram-negative bacteria. Suppression of Smad3 expression in mast cells may thus have therapeutic potential for Gram-negative bacterial infection such as acute septic peritonitis by augmenting innate immune responses of mast cells.