The use of prebiotics has an effect on postprandial glucose and insulin concentrations; however, the interaction between the previous profile of the intestinal microbiota and the effect of supplementation with prebiotics is not clear. Our objective was to evaluate the effect of previous intestinal microbiota profile on the postprandial insulin response to yacon syrup, used as a source of fructooligosaccharides (FOSs), in young women. The product presents high levels of FOS. In this double-blind, crossover, randomized clinical trial, 40 adult women were allocated to receive a breakfast containing 40 g of yacon syrup (14 g FOS, intervention A) or a breakfast containing 40 g of placebo (intervention B). On each intervention day, after 12 h of fasting, an aliquot of blood was collected for insulin analysis at 0, 15, 30, 45, 60, 90, and 120 min. The fecal sample was collected before the beginning of the interventions, and the DNA was extracted and quantified, with subsequent amplification of the 16S region, next-generation sequencing, and analysis of sequencing data. The glucose and insulin concentrations were reduced after ingestion of yacon syrup compared with placebo, specifically at the 30 min to insulin. After the receiver operating characteristic analysis, six volunteers who did not respond to the yacon consumption intervention were identified. The abundance of the phylum Actinobacteria (P = 0.021) and the order Bifidobacteriales (P = 0.013) were positively associated with better insulin response. Other main phyla were not associated with intervention response. The previous profile of the intestinal microbiota has an effect on the postprandial insulin response to FOSs, mainly in the phylum Actinobacteria and Bifidobacteriales order. Copyright © 2022 Elsevier Inc. All rights reserved.