Enteral feeding with small amounts to stimulate bowel motility, and glutamine supplementation, which provides nutrients selectively used by intestinal epithelial cells, might preserve the gut mucosa during fasting. We evaluated the effects of the interaction between mechanical strain and glutamine supplementation in human Caco-2 intestinal epithelial cells, and pursued the finding of equivalent effects of L- and D-glutamine in Caco-2, HT-29, and primary malignant human colonocytes. Caco-2 cells were subjected to repetitive strain in media containing 2 mmol/L of L-glutamine and media supplemented with L- or D-glutamine. Proliferation was determined by automated cell counting. Differentiation and cellular production of L-glutamine were determined spectroscopically. Rhythmic deformation stimulated Caco-2 proliferation in a frequency-dependent manner. Maximal stimulation occurred at 10 cpm, consistent with in vivo frequencies of peristalsis and villous motility. Deformation at 10 cpm and L-glutamine supplementation from 2 to 5 mmol/L concentrations independently stimulated Caco-2 proliferation; the combination further increased proliferation. D-Glutamine supplementation yielded similar results, although with lesser potency. Furthermore, both L- and D-glutamine equivalently reduced Caco-2 dipeptidyl dipeptidase activity. The effects of each isoform were blocked by 1 to 3 mmol/L acivicin, a selective antagonist of glutamine metabolism. Indeed Caco-2 and HT-29 cells and primary malignant colonocytes each metabolized D-glutamine to L-glutamine. Glutamine supplementation in fasting patients might prove synergistic with stimulation of bowel motility by non-nutritive feeding, whereas tissue-specific variations in D-glutamine metabolism might facilitate selective nutripharmaceutical targeting of the gut mucosa.