Abstract Background SEG-1, a Barrett's-derived esophageal adenocarcinoma cell line, is not responsive to transforming growth factor beta (TGF-β) growth effects. We hypothesize that SEG-1 cells lack the tumor-suppressor gene Runt domain transcription factor 3 (RUNX3) and that its reinstatement can restore the antiproliferative and apoptotic effects of TGF-β. Methods RUNX3 expression was assessed by immunoblotting. SEG-1 cells were transfected with RUNX3 and treated with TGF-β. The effects of RUNX3 transfection on cell proliferation and apoptosis were determined. Smad-mediated TGF-β transcriptional activity was evaluated with the use of dual-luciferase assay. Results SEG-1 cells are not responsive to TGF-β. SEG-1 cells lack RUNX3 protein expression, while RUNX3 is highly expressed in normal human gastric and esophageal epithelium. Although the Smad-2 signaling is activated by TGF-β, SEG-1 cells lack Smad-mediated TGF-β transcriptional activity. In cells transfected with RUNX3, TGF-β acquired an antiproliferative effect and induced apoptosis ( P=.001). RUNX3 transfection, in the absence of TGF-β, had no effect on proliferation and apoptosis of SEG-1 cells. RUNX3 expression dramatically increases SMAD-mediated TGF-β–induced transcriptional activity when compared with controls ( P = .0001). Conclusions RUNX3 is not expressed in SEG-1 cells, while it is present in normal esophageal mucosa. RUNX3 is essential for the antiproliferative and apoptotic effects of TGF-β in SEG-1 cells and for the Smad-mediated transcriptional activity of TGF-β.