Polyamines are molecules involved in cell growth and differentiation and are produced by bacterial metabolism. However, their production and effects by the microbiota selected by fructooligosaccharides consumption are controversial. In this study, we investigated the influence of supplementation of fructooligosaccharides on the cecal polyamine production by the microflora selected, and its effect on gut maturation in newborn piglets. Twenty piglets were fed a control formula (n = 10) or a formula supplemented with fructooligosaccharides (8 g/l) (n = 10) for 13 days. Colony-forming unit’s count of cecal content was done in different media. Several intestinal development parameters were measured as well as the polyamine concentration in the cecal mucosa and cecal content. A dose-dependent study on in vitro polyamine production by fructooligosaccharides addition to the isolated cecal content was performed. Bifidogenic activity of fructooligosaccharides increased polyamine concentration in the cecal content, mainly putrescine, with no beneficial effect on gut maturation. Bifidobacterium spp. were able to produce polyamines, but they were not the most significant bacterial producer of polyamines in the cecum of piglets fed fructooligosaccharides. Bifidogenic activity of fructooligosaccharides did not lead to an increase in gut maturation in piglets of 15 days of age although polyamines were increased in the cecal content.