BACKGROUND: Galacto-oligosaccharides (GOS) are non-digestible oligosaccharides with short galactosyl chain units produced by lactose fermentation which are considered as prebiotics. Only few studies have investigated the effects of GOS medium-term ingestion on the small intestinal epithelium characteristics. AIM OF THE STUDY: In this study, we evaluated the consequences of GOS ingestion on small intestinal mucosal morphology, on brush-border membrane enzyme activities and on mucin content in BALB/c mice. METHODS: Mice received the experimental diets for 4 weeks and then the small intestine was collected to measure sucrase, lactase and alkaline phosphatase activities, to study the villus heights in the jejunum mucosa and to determine mucosal mucin content as well as MUC-2 and MUC-4 mRNAs expression by qRT-PCR. RESULTS: Our results showed that GOS has no detectable effect on the intestine villus height but increased the total protein content by twofold. Sucrase activity was significantly increased in the intestinal mucosa recovered from animals fed the GOS diet without any detectable modification of lactase and phosphatase activities. Interestingly, GOS was also able to increase sucrase activity in cultured Caco-2 cells raising the view that they likely act directly on these cells. Furthermore, GOS was found to markedly increase O-linked glycoproteins associated with the intestinal mucosa without modifying MUC-2, MUC-4 mRNAs expression. Lastly, TNF-alpha mRNA expression was also not modified after GOS ingestion. CONCLUSIONS: These results suggest that, in BALB/c mice, 4-week GOS ingestion is able to increase the small intestinal mucosa-associated mucin content and enterocyte-associated sucrase activity without modifying villus height.