Emerging knowledge shows the importance of early life events in programming the intestinal mucosal immune system and development of the intestinal barrier function. These processes depend heavily on close interactions between gut microbiota and host cells in the intestinal mucosa. In turn, development of the intestinal microbiota is largely dependent on available nutrients and substrates required for the specific microbial community structures to expand. It is currently not known what the specificities are of intestinal microbial community structures in relation to the programming of the intestinal mucosal immune system and development of the intestinal barrier function. The objective of the present study was to investigate the effect of a nutritional intervention on intestinal development of suckling piglets by daily oral administration of fructooligosaccharides (FOS) over a period of 12 days. At the microbiota community level a clear “bifidogenic” effect of the FOS administration was observed in colon digesta at day 14. The former, however, did not translate into significant changes of local gene expression in the colonic mucosa. In the jejunum, significant changes were observed for microbiota composition at day 14, and microbiota diversity at day 25. In addition, significant differentially expressed gene sets in mucosal tissues of jejunum were identified at both days 14 and 25 of age. At the age of 14 days, lower activity of cell cycle-related processes and a higher activity of extracellular matrix processes were observed in jejunal scrapings of piglets supplemented with FOS compared to control piglets. At day 25, lower activity of immune-related processes in jejunal tissue were seen in piglets supplemented with FOS. Histological parameters, villi height and crypt depth, were significantly different at day 25 between the experimental and control group, where piglets supplemented with FOS had higher villi and deeper crypts. We conclude that oral FOS administration during the suckling period of piglets has significant bifidogenic effects on the microbiota in the colon and on gene expression in jejunal mucosa scrapings. We hypothesize that FOS supplementation of suckling piglets results in a higher butyrate production in the colon due to the increase in bifidobacteria and lactobacilli in the hindgut. We further speculate that a higher butyrate production in colonic digesta relates to changes in gene expression in the jejunum by thus far unknown mechanisms.