Background Severe Clostridium difficile toxin-induced enteritis is characterized by exuberant intestinal tissue inflammation, epithelial disruption and diarrhea. Adenosine, through its action on the adenosine A2A receptor, prevents neutrophillic adhesion and oxidative burst and inhibits inflammatory cytokine production. Alanyl-glutamine enhances intestinal mucosal repair and decreases apoptosis of enterocytes. This study investigates the protection from enteritis by combination therapy with ATL 370, an adenosine A2A receptor agonist, and alanyl-glutamine in a rabbit and murine intestinal loop models of C. difficile toxin A-induced epithelial injury. Methods Toxin A with or without alanyl-glutamine was administered intraluminally to rabbit ileal or murine cecal loops. Animals were also given either PBS or ATL 370 parenterally. Ileal tissues were examined for secretion, histopathology, apoptosis, Cxcl1/KC and IL-10. Results ATL 370 decreased ileal secretion and histopathologic changes in loops treated with Toxin A. These effects were reversed by the A2A receptor antagonist, SCH 58261, in a dose-dependent manner. The combination of ATL 370 and alanyl-glutamine significantly further decreased ileal secretion, mucosal injury and apoptosis more than loops treated with either drug alone. ATL 370 and alanyl-glutamine also decreased intestinal tissue KC and IL-10. Conclusions Combination therapy with an adenosine A2A receptor agonist and alanyl-glutamine is effective in reversing C. difficile toxin A-induced epithelial injury, inflammation, secretion and apoptosis in animals and has therapeutic potential for the management of C. difficile infection.