Genes involved in the transforming growth factor beta (TGF-beta) signaling pathway are frequently altered in several types of cancers, and a gastric tumor suppressor RUAW3 appears to be an integral component of this pathway. We reported previously that apoptosis is notably reduced in Runx3(-/-) gastric epithelial cells. In the present study, we show that a proapoptotic gene Bim was transcriptionally activated by RUNX3 in the gastric cancer cell lines SNU16 and SNU719 treated with TGF-beta. The human Bim promoter contains RUNX sites, which are required for its activation. Furthermore, a dominant negative form of RUNX3 comprised of amino acids I to 187 increased tumorigenicity of SNU16 by inhibiting Bim expression. In Runx3(-/-) mouse gastric epithelium, Bim was down-regulated, and apoptosis was reduced to the same extent as that in Bim(-/-) gastric epithelium. We confirmed comparable expression of TGF-beta I and TGF-beta receptors between wild-type and Runx3(-/-) gastric epithelia and reduction of Bim in TGF-beta 1(-/-) stomach. These results demonstrate that RUNX3 is responsible for transcriptional up-regulation of Bim in TGF-beta-induced apoptosis.