The aim of this study was to investigate the effect of Clostridium difficile toxin A (TxA) on intestinal epithelial cell migration, apoptosis, and transepithelial resistance and to evaluate the effect of glutamine (Gln) and its stable derivative, alanyl-glutamine (Ala-Gln), on TxA-induced damage. Migration was measured in rat intestinal epithelial cells (IEC-6) 6 and 24 hr after a razor scrape of the cell monolayer. Cell proliferation was indirectly measured utilizing the tetrazolium salt WST-1. The cells were incubated with TxA (1-100 ng/ml) in medium without Gln or medium containing Gln or Ala-Gln (1-30 mM). Apoptosis was quantified in IEC-6 cells using annexin V assay. Transepithelial resistance was measured using an epithelial voltohmmeter across T84 cells seeded on a transwell filter. TxA-induced a dose-dependent reduction of migration and also caused dose and time-dependent apoptosis in IEC-6 cells. Gln and Aln-Gln significantly enhanced IEC-6 cell migration and proliferation. Gln and Ala-Gln also prevented the inhibition of migration, apoptosis, and the initial drop in transepithelial resistance induced by TxA. In conclusion, both peptides reduced toxin-induced epithelial damage and thus might play an adjunctive role in C. difficile-induced colitis therapy.