Apoptosis signal-regulating kinase-1 (ASK-1) is an important molecule for the pro-apoptotic signaling. ASK-1 also contributes to the cellular survival for many types of cells. Thus, ASK-1 has a broad range of biological activities depending on the cell type. The present study assessed the role(s) of ASK-1 in colorectal cancer cells (HT-29) by using adenovirus vectors expressing wild-type (WT)-ASK-1 or dominant-negative (DN) mutant of ASK-1 and recombinant adenovirus containing the bacterial beta-galactosidase gene (Ad-LacZ), a negative control for Ad-DN-ASK-1. Selective phosphorylation of ASK-1 at Thr 845, a kinase domain site, but not Ser 83 nor 967 sites was induced by serum stimulation in a time-dependent manner. Transfection with Ad-DN-ASK-1 inhibited the serum-induced phosphorylation of p38 mitogen-activated protein kinase, a downstream molecule of ASK-1. Transfection with Ad-DN-ASK-1 diminished the serum-induced cell proliferation in a dose-dependent manner, whereas WT-ASK-1 increased it. Apoptosis assessed by Hoechst staining was induced in the Ad-DN-ASK-1 treated cells. In vivo transfection of Ad-DN-ASK-1 into tumor xenografts of HT-29 cells in nude mice significantly decreased the tumor volume on day 29. Cleaved caspase-3 was found in the tumors of DN-ASK-1 treated mice. We obtained the first evidence that DN-ASK-1 transfection exerted significant antitumor effects on colon cancer mediated by apoptosis.