Glutamine, the most abundant amino acid in the bloodstream, is the preferred fuel source for enterocytes and plays a vital role in the maintenance of mucosal growth. The molecular mechanisms regulating the effects of glutamine on intestinal cell growth and survival are poorly understood. Here, we show that addition of glutamine (1 mmol/L) enhanced rat intestinal epithelial (RIE-1) cell growth; conversely, glutamine deprivation increased apoptosis as noted by increased DNA fragmentation and caspase-3 activity. To delineate signaling pathways involved in the effects of glutamine on intestinal cells, we assessed activation of extracellular signal-related kinase (ERK), protein kinase D (PKD) and phosphatidylinositol 3-kinase (PI3K)/Akt, which are important pathways in cell growth and survival. Addition of glutamine activated ERK and PKD in RIE-1 cells after a period of glutamine starvation; inhibition of ERK, but not PKD, increased cell apoptosis. Conversely, glutamine starvation alone increased phosphorylated Akt; inhibition of Akt enhanced RIE-1 cell DNA fragmentation. The role of ERK was further delineated using RIE-1 cells stably transfected with an inducible Ras. Apoptosis was significantly increased following ERK inhibition despite Ras activation. Taken together, these results identify a critical role for the ERK signaling pathways in glutamine-mediated intestinal homeostasis. Furthermore, activation of PI3K/Akt during periods of glutamine deprivation likely occurs as a protective mechanism to limit apoptosis associated with cellular stress. Importantly, our findings provide novel mechanistic insights into the anti-apoptotic effects of glutamine in the intestine.