Glutamine (Gln) is the most concentrated amino acid in blood and considered conditionally essential. Its requirement is increased during physiological stress, such as malnutrition or illness, despite its production by muscle and other organs. In the malnourished state Gln has been suggested to have a trophic effect on the exocrine pancreas and small intestine. However, the Gln transport capacity, the functional relationship of these two organs and the potential role of the Gln-glutamate (Glu) cycle are unknown. We observed that pancreatic acinar cells express lower levels of Glu than Gln transporters. Consistent with this expression pattern the rate of Glu influx into acinar cells was approximately 6-fold lower than that of Gln. During protein restriction, acinar cell glutaminase expression was increased and Gln accumulation maintained. Moreover, Glu secretion by acinar cells into pancreatic juice and thus into the lumen of the small intestine was maintained. In the intestinal lumen, Glu absorption was preserved and glutamate dehydrogenase expression was augmented, potentially providing the substrates for increasing energy production via the TCA cycle. Our findings suggest that one mechanism by which Gln exerts a positive effect on exocrine pancreas and small intestine involves the Gln metabolism in acinar cells and the secretion of Glu into the small intestine lumen. The exocrine pancreas acinar cells not only avidly accumulate Gln; but metabolize Gln to generate energy and to synthesize Glu for secretion in the pancreatic juice. Secreted Glu is suggested to play an important role during malnourishment in sustaining small intestinal homeostasis.