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Glutamine is an essential amino acid for enterocytes, especially in states of critical illness and injury. In several studies it has been speculated that the beneficial effects of glutamine are dependent on the route of supply (luminal or systemic). The aim of this study was to investigate the relevance of both routes of glutamine delivery to in vitro intestinal cells and to explore the molecular basis for proposed beneficial glutamine effects: (a) by determining the relative uptake of radiolabelled glutamine in Caco-2 cells; (b) by assessing the effect of glutamine on the proteome of Caco-2 cells using a 2D gel electrophoresis approach; and (c) by examining glutamine incorporation into cellular proteins using a new mass spectrometry-based method with stable isotope labelled glutamine. Results of this study show that exogenous glutamine is taken up by Caco-2 cells from both the apical and the basolateral side. Basolateral uptake consistently exceeds apical uptake and this phenomenon is more pronounced in 5-day-differentiated cells than in 15-day-differentiated cells. No effect of exogenous glutamine supply on the proteome was detected. However, we demonstrated that exogenous glutamine is incorporated into newly synthesized proteins and this occurred at a faster rate from basolateral glutamine, which is in line with the uptake rates. Interestingly, a large number of rapidly labelled proteins is involved in establishing cell-cell interactions. In this respect, our data may point to a molecular basis for observed beneficial effects of glutamine on intestinal cells and support results from studies with critically ill patients where parenteral glutamine supplementation is preferred over luminal supplementation.