Bile acids (BAs) are key mediators of the glycemic control after bariatric surgeries. Cholecystectomy modifies the kinetics of BAs, and whether this procedure influences the BAs pool and its metabolic response to bariatric surgeries is not known. We used targeted and untargeted metabolomics to assess whether cholecystectomy influenced plasma and fecal BAs fluctuations and the systemic metabolomic profile after Roux-en-Y gastric bypass (RYGB). Women with obesity and type 2 diabetes were included. Sample collections and clinical evaluations were performed before and 3 months after RYGB. RYGB influenced 9 fecal and 3 plasma BAs in patients with cholecystectomy (p ≤ 0.05). Comparisons between patients with and without cholecystectomy revealed different concentrations of 4 fecal and 5 plasma BAs (p ≤ 0.05). Cholecystectomy impacted the global metabolomics responses to RYGB, and patients who underwent the gallbladder removal also lacked some significant improvements in clinical markers, primarily the lipid profile. By affecting the BAs concentrations, cholecystectomy seems to alter the systemic metabolic response to RYGB. Therefore, cholecystectomy may act as a bias in assessments of the metabolic effects of bariatric surgeries and their relationships with clinical outcomes.