We evaluated whether the excluded stomach (ES) after Roux-en-Y gastric bypass (RYGB) can represent a premalignant environment. Twenty obese women were prospectively submitted to double-balloon enteroscopy (DBE) with gastric juice and biopsy collection, before and 3 months after RYGB. We then evaluated morphological and molecular changes by combining endoscopic and histopathological analyses with an integrated untargeted metabolomics and transcriptomics multiplatform. Preoperatively, 16 women already presented with gastric histopathological alterations and an increased pH (≥4.0). These gastric abnormalities worsened after RYGB. A 90-fold increase in the concentration of bile acids was found in ES fluid, which also contained other metabolites commonly found in the intestinal environment, urine, and faeces. In addition, 135 genes were differentially expressed in ES tissue. Combined analysis of metabolic and gene expression data suggested that RYGB promoted activation of biological processes involved in local inflammation, bacteria overgrowth, and cell proliferation sustained by genes involved in carcinogenesis. Accumulated fluid in the ES appears to behave as a potential premalignant environment due to worsening inflammation and changing gene expression patterns that are favorable to the development of cancer. Considering that ES may remain for the rest of the patient's life, long-term ES monitoring is therefore recommended for patients undergoing RYGB.