CUGBP2 is a RNA binding protein that regulates mRNA translation and modulates apoptosis. Here, we report the identification of two splice variants (termed variants 2 and 3) in cultured human intestinal epithelial cells and in mouse gastrointestinal tract. The variants are generated from alternative upstream promoters resulting in the inclusion of additional N-terminal residues. While variant 2 is the predominant isoform in normal intestine, its expression is reduced while variant 1 is overexpressed following -irradiation. All three variants bind cyclooxygenase-2 (COX-2) mRNA. However, only variant 1 inhibits the translation of the endogenous COX-2 mRNA and a chimeric luciferase mRNA containing the COX-2 3'UTR. Furthermore, while variant 1 is predominantly nuclear, variants 2 and 3 are predominantly cytoplasmic. These data imply that the additional amino acids affect CUGBP2 function. Previous studies have demonstrated that variant 1 induces intestinal epithelial cells to undergo apoptosis. However, in contrast to variant 1, the two novel variants do not affect proliferation or apoptosis of HCT116 cells. In addition, only variant 1 induced G2/M cell cycle arrest, which was overcome by prostaglandin E2 (PGE2). Moreover, variant 1 increased cellular levels of phosphorylated p53 and Bax and decreased Bcl2. Caspase-3 and -9 were also activated suggesting the initiation of the intrinsic apoptotic pathway. Furthermore, increased phosphorylation of Chk1 and Chk2 kinases, and increased nuclear localization of Cdc2 and cyclin B1 suggested that cells were in mitotic transition. Taken together, these data demonstrate that cells expressing CUGBP2 variant 1 undergo apoptosis during mitosis suggesting mitotic catastrophe.