Renewal of the gastrointestinal epithelium involves a coordinated process of terminal differentiation and programmed cell death. Integrins have been implicated in the control of apoptotic processes in various cell types. Here we examine the role of integrins in the regulation of apoptosis in gastrointestinal epithelial cells with the use of a rat small intestinal epithelial cell line (RIE1) as a model. Overexpression of the integrin α5 subunit in RIE1 cells conferred protection against several proapoptotic stimuli. In contrast, overexpression of the integrin α2 subunit had no effect on cell survival. The antiapoptotic effect of the α5 subunit was partially retained by a mutated version that had a truncation of the cytoplasmic domain. The antiapoptotic effects of the full-length or truncated α5 subunit were reversed upon treatment with inhibitors of phosphatidylinositol 3-kinase (PI-3-kinase), suggesting that the α5β1 integrin might interact with the PI-3-kinase/Akt survival pathway. When cells overexpressing α5 were allowed to adhere to fibronectin, there was a moderate activation of protein kinase B (PKB)/Akt, whereas no such effect was seen in α2-overexpressing cells adhering to collagen. Furthermore, in cells overexpressing α5 and adhering to fibronectin, there was a dramatic enhancement of the ability of growth factors to stimulate PKB/Akt; again, this was not seen in cells overexpressing α2 subunit and adhering to collagen or fibronectin. Expression of a dominant negative version of PKB/Akt in RIE cells blocked to ability of α5 to enhance cell survival. Thus, the α5β1 integrin seems to protect intestinal epithelial cells against proapoptotic stimuli by selectively enhancing the activity of the PI-3-kinase/Akt survival pathway.