Obesity is associated with an increase in the fractional contribution of gluconeogenesis (GNG) to glucose production. We tested if this was related to the altered protein metabolism in obesity. GNG(PEP) (via phosphoenol pyruvate [PEP]) was measured after a 17-h fast using the deuterated water method and 2H nuclear magnetic resonance spectroscopy of plasma glucose. Whole-body 13C-leucine and 3H-glucose kinetics were measured in the postabsorptive state and during a hyperinsulinemic-euglycemic-isoaminoacidemic clamp in 19 (10 men and 9 women) lean and 16 (7 men and 9 women) obese nondiabetic subjects. Endogenous glucose production was not different between groups. Postabsorptive %GNG(PEP) and GNG(PEP) flux were higher in obese subjects, and glycogenolysis contributed less to glucose production than in lean subjects. GNG(PEP) flux correlated with all indexes of adiposity and with postabsorptive leucine rate of appearance (Ra) (protein catabolism). GNG(PEP) was negatively related to the clamp glucose rate of disposal (Rd) and to the protein anabolic response to hyperinsulinemia. In conclusion, the increased contribution of GNG to glucose production in obesity is linked to increased postabsorptive protein catabolism and insulin resistance of both glucose and protein metabolism. Due to increased protein turnover rates, greater supply of gluconeogenic amino acids to the liver may trigger their preferential use over glycogen for glucose production.