Glutamine (Gln), the most abundant amino acid in the bloodstream, is essential for maintenance of gut homeostasis and proliferation. Deprivation of Gln induces intestinal cell apoptosis and mucosal atrophy; however, the molecular mechanisms regulating the effects of Gln are poorly understood. Protein kinase D (PKD), a novel protein kinase which is structurally distinct from PKC proteins, has been implicated in numerous cellular functions including cell proliferation and anti-apoptotic signals. The purpose of our study was to determine whether Gln activates PKD which ultimately enhances intestinal cell survival. Methods: (i) Rat intestinal epithelial (RIE-1) cells were maintained in the presence or absence of Gln for 24 h; cell survival and apoptosis were determined by MTT assay and Cell Death ELISA, respectively. (ii) RIE cells were serum and Gln deprived for 24 h. Cells were then treated with Gln (1 mmol/L) or vehicle (control); cells were lysed and protein extracted over a time course (0, 1, 2, 4, 18, and 24 h). Expression of phosphorylated and total PKD was assessed by Western blot. Results: Gln deprivation significantly reduced cell survival when compared to cells maintained in the presence of Gln (p<0.05). DNA fragmentation, an indicator of apoptosis, was increased in cells deprived of Gln although this did not achieve statistical significance. Interestingly, phosphorylated (i.e. activated) PKD levels were increased at 1, 2, 4, 18, and 24 h after addition of Gln; total PKD expression was not affected. Conclusions: Gln is an essential amino acid for intestinal cell growth and survival. We demonstrate, for the first time, that addition of Gln increases expression of phosphorylated PKD in intestinal cells; this induction of activated PKD likely contributes to the cell survival effects of Gln. Moreover, our results identify a critical role for the PKD protein in gut homeostasis.