One-anastomosis gastric bypass (OAGB) has gained importance as a safe and effective operation to treat morbid obesity. It is not known whether a long biliopancreatic limb (BPL) in OAGB surgery compared to a short BPL results in beneficial metabolic outcomes. 5-week-old male C57BL/6J mice fed a high-fat diet for 8 weeks underwent OAGB surgery with defined short and long BPL lengths, or sham surgery combined with caloric restriction. Weight loss, glucose tolerance, obesity-related comorbidities, endocrine effects, gut microbiota and bile acids were assessed. Total weight loss was independent of the length of the BPL after OAGB surgery. However, a long BPL was associated with lower glucose-stimulated insulin on day 14, and an improved glucose tolerance on day 35 after surgery. A long BPL resulted in reduced total cholesterol. There were no differences in the resolution of metabolic dysfunction-associated steatotic liver disease and adipose tissue inflammation. Tendencies of an attenuated hypothalamic-pituitary-adrenal axis and aldosterone were present in the long BPL group. In OAGB-operated mice, we found an increase in primary conjugated bile acids (pronounced in long BPL) along with a loss in bacterial Desulfovibrionaceae and Erysipelotrichaceae and simultaneous increase in Akkermansiaceae, Sutterellaceae and Enterobacteriaceae. In sum, OAGB surgery with a long compared to a short BPL led to similar weight loss, but improved glucose metabolism, lipid and endocrine outcomes in obese mice, potentially mediated through changes in gut microbiota and related bile acids. Tailoring the BPL length in humans might help to optimize metabolic outcomes after bariatric surgery.