BACKGROUND: The aim of the study was to investigate the role of the gut microbiota in weight regain or suboptimal weight loss following Roux-en-Y gastric bypass (RYGB). MATERIALS AND METHODS: The gut microbiota composition in post-RYGB patients who experienced successful weight loss (SWL, n = 6), post-RYGB patients who experienced poor weight loss (PWL, n = 6), and non-surgical controls (NSC, n = 6) who were age- and BMI-matched to the SWL group (NSC, n = 6) were characterized through 16S rRNA gene sequencing. To further investigate the impact of the gut microbiota on weight profile, human fecal samples were transplanted into antibiotic-treated mice. RESULTS: Orders of Micrococcales and Lactobacillales were enriched in SWL and PWL groups compared to the NSC group. No significant difference was observed in the gut microbiota composition between PWL and SWL patients. However, transfer of the gut microbiota from human patients into antibiotic-treated mice resulted in significantly greater weight gain in PWL recipient mice compared to SWL recipient mice. A few genera that were effectively transferred from humans to mice were associated with weight gain in mice. Among them, Barnesiella was significantly higher in PWL recipient mice compared to SWL and NSC recipient mice. CONCLUSION: These results indicate that the gut microbiota are at least functionally, if not compositionally, different between PWL and SWL patients. Some taxa may contribute to weight gain after surgery. Future studies will need to determine the molecular mechanisms behind the effects of the gut bacteria on weight regain after RYGB.