Aims: Isomalto-oligosaccharides (IMO) with alpha(1 -> 6) and alpha(1 -> 4) glucosidic linkages are produced by enzymatic conversion of starch. IMO are only partially digestible but data on their influence on intestinal microbiota are limited. It was the aim of this study to investigate the effect of IMO diet on intestinal microbiota and short-chain fatty acids production (SCFA) in rats. Methods and results: Three groups of F344 rats, each consisting of six animals, were fed IMO, inulin or a control diets for six weeks. A qualitative assessment of the intestinal microbiota was achieved by PCR-denaturing gradient gel electrophoresis (DGGE). Major bacterial taxa were quantified by quantitative PCR (qPCR), and SCFA were measured using gas chromatography. Quantitative PCR demonstrated that lactobacilli were one of the dominant bacterial taxa in faecal samples from rats. IMO increased the number of lactobacilli and the total number of intestinal bacteria in rats fed IMO compared with animals receiving control and inulin diets. Furthermore, PCR-DGGE with lactobacilli-specific primers showed an altered biodiversity of lactobacilli in rats fed IMO compared with control diet. Conclusions: IMO selectively stimulates lactobacilli and increases their diversity in rats. Significance and impact of study: Isomalto-oligosaccharides specifically stimulate growth of intestinal lactobacilli in a rat model system.