Prebiotic fibers may alter gastrointestinal (GI) transit time, microbiota composition and short chain fatty acid (SCFA) production, contributing to improved gut functionality and metabolic health. We investigated long-term effects of Arabinoxylan-Oligosaccharide (AXOS), a prebiotic dietary fiber on GI transit time, gut microbiota composition, and metabolic profile in adult participants.Methods: This randomized, placebo-controlled double-blind parallel study included 48 normoglycemic men and women (ages 20-55 y, body mass index (BMI) 19.8-30.5 kg/m2) with a slow whole-gut transit time (>35h) recruited during August 2015 to December 2016 in Maastricht, the Netherlands. Participants were randomly allocated to 12 weeks 15g/day AXOS or placebo (maltodextrin) intake. GI transit time, stool parameters, gut permeability, SCFA and microbiota composition were assessed before and after. Energy expenditure, substrate oxidation, glucose, insulin, lipids and incretin hormones were measured during a breakfast meal test before and after.Results: AXOS significantly changed the microbiota (p=0.05) mainly by increasing Bifidobacterium and decreased microbial alpha-diversity (P<.001) as compared to placebo. Whole-gut and upper intestinal transit were not affected, but stool consistency softened after AXOS (Bristol stool chart score 2.7 ± 0.19 to 3.3 ± 0.19, P<.01). Postprandial fat oxidation tended to increase (iAUC, P=.073) and early GLP-1 response (AUC0-90min, P=.005) was reduced after AXOS. Energy expenditure, plasma metabolites, SCFA concentrations and gut permeability were unchanged. Microbiota could classify responders in improved whole-gut transit after AXOS with an ([ROC] AUC 0.80%).Conclusion: AXOS intake, changed the microbiota, mainly increased fecal Bifidobacterium, tended to increase postprandial fat oxidation and decreased the early GLP-1 response. Whilst we did not observe changes in whole-gut transit time, overall microbiota could accurately classify responders with improved GI transit after AXOS intake.