Roux-en-Y Gastric Bypass (RYGB) surgery is one of the most effective approaches for the treatment of severe obesity. Despite substantial weight loss following RYGB, a considerable proportion of patients experience weight regain or insufficient weight loss. The proposed research aimed to investigate the role of the gut microbiota in weight regain or suboptimal weight loss following RYGB. 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 through oral gavage. Food intake and body weight were measured at weekly intervals for a month. At five weeks following colonization mice were randomly switched to a Western Diet or maintained on a normal diet. The results showed that Lactobacillales, Enterobacteriales, and Verrucomicrobials were enriched in both surgical 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 at four weeks following colonization (15.03±2.59% versus 7.88±1.28%, F(2,41)=4.01 p=0.026). We found that Barnesiella, Gordonibacter, Parasutterella, Clostridium cluster XVIa were effectively transferred from humans to mice and were associated with weight gain in recipient mice. Interestingly, Barnesiella that tended to be higher in PWL humans was also significantly higher in PWL recipient mice compared to SWL and NSC recipient mice. All three groups of recipient mice gained weight when they were placed on the Western Diet regardless of human donor group. In summary, the results indicate that the gut microbiota are at least functionally 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.