D-Tagatose is partly absorbed in the stomach and small intestine. Most of it is fermented by the large intestinal microbiota. The effect of D-tagatose on the composition of the microbiota and production of short chain fatty acids (SCFAs) was studied in vivo and in vitro. Gastrointestinal (GI) complaints were also studied. The in vivo study was performed according to a randomized, placebo-controlled, double-blind, five-way cross-over design in healthy subjects (12 men and 18 women). All subjects consumed 30 g raspberry jam containing 7.5 or 12.5 g D-tagatose, 7.8 g fructo-oligosaccharides (positive reference), 7.6 g D-tagatose plus 7.5 g fructo-oligosaccharides, or 15.1 g sucrose (negative reference) at breakfast for 2 weeks in different orders. At the end of each treatment period lipids and safety parameters in blood and GI complaints were evaluated by questionnaires, and faecal microbiota and SCFAs were measured. Furthermore, test-tube incubations of faecal slurries with D-tagatose, fructo-oligosaccharides and sucrose were performed. An in vitro model simulating the large intestine was used to assess the mechanistic effect of D-tagatose on microbiota composition and SCFA production. The high-tagatose treatment resulted in increased numbers of faecal lactobacilli in men, but not in women. Also in vitro, lactobacilli increased. Both the test-tube incubations of fresh faeces from the in vivo study with D-tagatose and the study in the in vitro model showed increased butyrate production after all treatments with D-tagatose. High-tagatose, but not low-tagatose, resulted in a slightly increased defecation frequency and stools of thinner consistency. Only a few GI complaints were reported. The data indicate that daily consumption of 7.5 or 12.5 g D-tagatose may lead to increased production of butyrate and to an increase of lactobacilli, without serious GI complaints. In view of the health-promoting effects of butyrate and lactobacilli, D-tagatose may be considered a prebiotic substrate. © 2005 Taylor & Francis Group Ltd.