The gut microbiota plays an important role in human health by interacting with host diet, but there is substantial inter-individual variation in the response to diet. Here we compared the gut microbiota composition of healthy subjects who exhibited improved glucose metabolism following 3-day consumption of barley kernel-based bread (BKB) with those who responded least to this dietary intervention. The Prevotella/Bacteroides ratio was higher in responders than non-responders after BKB. Metagenomic analysis showed that the gut microbiota of responders was enriched in Prevotella copri and had increased potential to ferment complex polysaccharides after BKB. Finally, germ-free mice transplanted with microbiota from responder human donors exhibited improved glucose metabolism and increased abundance of Prevotella and liver glycogen content compared with germ-free mice that received non-responder microbiota. Our findings indicate that Prevotella plays a role in the BKB-induced improvement in glucose metabolism observed in certain individuals, potentially by promoting increased glycogen storage. Display omitted •Prevotella/Bacteroides is associated with a beneficial response to barley kernels•Prevotella-enriched microbial interactions are higher in barley kernel responders•Prevotella protects against Bacteroides-induced glucose intolerance•Prevotella promotes increased hepatic glycogen storage in mice Diet affects the gut microbiota composition, though large inter-individual variations exist. Kovatcheva-Datchary et al. reveal that subjects with improved glucose metabolism after barley kernel supplementation have increased Prevotella in their gut microbiota. Prevotella plays a direct role in the beneficial response, supporting the importance of personalized approaches to improve metabolism.