The gut microbiota might be a critical modifier of metabolic disease development. Dietary fibers such as galacto-oligosaccharides (GOS) presumably stimulate the growth of bacteria beneficial for metabolic health. This study aimed to assess the impact of GOS on obesity, glucose and lipid metabolism.Following Western-type diet feeding (C57BL/6 mice) with or without β-GOS (7% w/w, 15 weeks), body composition, glucose and insulin tolerance tests, lipid profiles, fat kinetics studies and microbiota analyses were performed. GOS reduced body weight gain (p<0.01), accumulation of epididymal (p<0.05) and perirenal (p<0.01) fat, and development of insulin resistance (p<0.01). GOS-fed mice had lower plasma cholesterol (p<0.05), mainly within low-density lipoproteins, lower intestinal fat absorption (p<0.01), more fecal neutral sterol excretion (p<0.05). and higher intestinal GLP-1 expression (p<0.01). Fecal bile acid excretion was lower (p<0.01) in GOS-fed mice with substantial compositional differences, namely decreased cholic (p<0.05), α-muricholic (p<0.05), and deoxycholic acid excretion (p<0.01), whereas hyodeoxycholic acid increased (p<0.01). Substantial changes in microbiota composition, conceivably beneficial for metabolic health occured upon GOS feeding.GOS supplementation to Western-type diet improved body weight gain, dyslipidemia and insulin sensitivity supporting a therapeutic potential of GOS for individuals at risk of developing metabolic syndrome. This article is protected by copyright. All rights reserved.