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Although bariatric surgery is known to change the metabolome, it is unclear if this is specific for the intervention or a consequence of the induced bodyweight loss. As the weight loss after Roux-en-Y Gastric Bypass (RYGB) can hardly be mimicked with an evenly effective diet in <i>humans</i>, translational research efforts might be helpful. A group of 188 plasma metabolites of 46 patients from the randomized controlled Würzburg Adipositas Study (WAS) and from RYGB-treated <i>rats</i> (<i>n</i> = 6) as well as body-weight-matched controls (<i>n</i> = 7) were measured using liquid chromatography tandem mass spectrometry. WAS participants were randomized into intensive lifestyle modification (LS, <i>n</i> = 24) or RYGB (OP, <i>n</i> = 22). In patients in the WAS cohort, only bariatric surgery achieved a sustained weight loss (BMI −34.3% (OP) vs. −1.2% (LS), <i>p</i> ≤ 0.01). An explicit shift in the metabolomic profile was found in 57 metabolites in the <i>human</i> cohort and in 62 metabolites in the rodent model. Significantly higher levels of sphingolipids and lecithins were detected in both surgical groups but not in the conservatively treated <i>human</i> and animal groups. RYGB leads to a characteristic metabolomic profile, which differs distinctly from that following non-surgical intervention. Analysis of the <i>human</i> and <i>rat</i> data revealed that RYGB induces specific changes in the metabolome independent of weight loss.