Background <p>Overweight increases the risk of Type 2 Diabetes (T2D), and insulin resistance (IR) precedes development of T2D. Besides lifestyle factors, gut microbes and their metabolites contribute to IR. Gut microbiota could be modulated by probiotics and prebiotics to improve IR, but current research results from interventions are controversial. We aimed at exploring the effects of prebiotic xylo-oligosaccharides (XOS) on the structure and metabolites of gut microbiota, IR, body composition and inflammatory response in humans with overweight.</p> Methods <p>In this within-subjects study design the participants had a 1-month baseline control period without changing their lifestyle and then, they were subjected to 4-months of dietary XOS supplementation. Based on baseline HOMA-IR, we divided study participants into insulin sensitive (&lt; 3, IS, <i>n</i> = 11) and insulin resistant (&gt; 3, IR, <i>n</i> = 38) groups. The gut microbiota were analyzed using 16&#xa0;S rRNA gene sequencing. Fecal metabolomes were quantified using nuclear magnetic resonance spectroscopy (NMR). Additionally, blood clinical variables, serum cytokines, body composition and diet were studied.</p> Results <p>We identified clear differences between insulin-resistant (IR) and insulin-sensitive (IS) participants in gut microbiome, body composition, and glucose–insulin profiles, along with modest variations in proinflammatory cytokines. Although four months of XOS supplementation did not alter insulin sensitivity or most other measured outcomes, it slightly reduced the levels of total cholesterol and increased HDL cholesterol in IR participants. However, it should be noted that during control period HDL first decreased in that group. Interestingly, XOS treatment decreased fecal galactose, isobutyrate, and isovalerate levels in IS participants, whereas shifts in microbial abundances (20 different microbial taxa) were observed only in IR participants with no changes in diversity. Isobutyrate, isovalerate and phenylacetate levels were lower in the IR group at baseline compared to IS. Across the study from baseline to post-treatment, body weight and BMI declined in IR individuals.</p> Conclusions <p>XOS supplementation altered gut microbiota composition in IR participants, while changing fecal metabolome in IS participants. XOS modestly improved lipid parameters, such as HDL cholesterol, but did not improve insulin sensitivity, body composition, or inflammatory markers. More and larger studies are needed to show that prebiotic XOS can consistently improve insulin sensitivity.</p> Trial registration <p>The study was retrospectively registered at ISRCTN under the number ISRCTN86495943.</p>