Amino acids interact with glucose metabolism both as carbon substrates and by recycling glucose carbon via alanine and glutamine; however, the effect of protein intake on glucose homeostasis during weight loss remains unknown. This study tests the hypothesis that a moderate increase in dietary protein with a corresponding reduction of carbohydrates (CHO) stabilizes fasting and postprandial blood glucose and insulin during weight loss. Adult women (n = 24; >15% above ideal body weight) were assigned to either a Protein Group [protein: 1.6 g/(kg. d); CHO <40% of energy] or CHO Group [protein: 0.8 g/(kg. d); CHO >55%]. Diets were equal in energy (7100 kJ/d) and fat (50 g/d). After 10 wk, the Protein Group lost 7.53 +/- 1.44 kg and the CHO Group lost 6.96 +/- 1.36 kg. Plasma amino acids, glucose and insulin were determined after a 12-h fast and 2 h after a 1.67 MJ test meal containing either 39 g CHO, 33 g protein and 13 g fat (Protein Group) or 57 g CHO, 12 g protein and 14 g fat (CHO Group). After 10 wk, subjects in the CHO Group had lower fasting (4.34 +/- 0.10 vs 4.89 +/- 0.11 mmol/L) and postprandial blood glucose (3.77 +/- 0.14 vs. 4.33 +/- 0.15 mmol/L) and an elevated insulin response to meals (207 +/- 21 vs. 75 +/- 18 pmol/L). This study demonstrates that consumption of a diet with increased protein and a reduced CHO/protein ratio stabilizes blood glucose during nonabsorptive periods and reduces the postprandial insulin response.