Background/aims: The effects of short-chain fatty acids and glutamine on diseased colonic mucosa, such as in inflammatory bowel disease, are not well described. The aim of this study was to investigate the role of L-glutamine and short-chain fatty acids, both via enema and oral administration, on mucosal healing in experimental colitis. Methods: Colitis was induced with trinitrobenzenesulphonic acid in ethanol enema in rats. Saline enema (Colitis group, n: 12), L-glutamine enema (n: 12), short-chain fatty acids enema (n: 12), oral L-glutamine (n:11) and oral short-chain fatty acids (n. 11) were applied twice daily for 10 days after induction of colitis. The sham group (n: 12) received only saline enema. Rats were sacrificed at the tenth day. Crypt depth and DNA content were measured in colonic mucosa. Results: Crypt depth was significantly greater in both glutamine groups and short-chain fatty acids enema group than in sham and colitis groups (p<0.05). The mucosal DNA contents of the colitis and glutamine enema groups were significantly greater than both short-chain fatty acids groups (p<0.05). DNA content in the oral glutamine group was significantly greater than in the short-chain fatty acids enema group (p<0.05). Conclusions: L-glutamine enema can accelerate mucosal healing and regeneration in experimentally induced colitis in rats. When compared to glutamine in this study, short-chain fatty acids showed no beneficial effect on colitis.