Background: Early-life nutrition has a programming effect on later metabolic health; however, the impact of exposure to a high-protein (HP) diet is still being investigated. Objective: This study evaluated the consequences on pup phenotype of an HP diet during gestation and lactation and after weaning. Methods: Wistar rat dams were separated into 2 groups fed an HP (55% protein) or normal protein (NP) (control; 20% protein) isocaloric diet during gestation, and each group subsequently was separated into 2 subgroups that were fed an HP or NP diet during lactation. After weaning, male and female pups from each mother subgroup were separated into 2 groups that were fed either an NP or HP diet until they were 6 wk old. Measurements included weight, food intake, body composition, blood glucose, insulin, glucagon, leptin, insulin-like growth factor I, and lipids. Results: Feeding mothers the HP diet during gestation or lactation induced lower postweaning pup weight (gestation diet x time, P \textless 0.0001; lactation diet x time, P \textless 0.0001). Regardless of dams' diets, pups receiving HP compared with NP diet after weaning had 7% lower weight (NP, 135.0 +/- 2.6 g; HP, 124.4 +/- 2.5 g; P \textless 0.0001), 16% lower total energy intake (NP, 777 +/- 14 kcal; HP, 649 13 kcal; P \textless 0.0001) and 31% lower adiposity (P \textless 0.0001). Pups receiving HP compared with NP diet after weaning had increased blood glucose, insulin, and glucagon when food deprived (P \textless 0.0001 for all). The HP compared with the NP diet during gestation induced higher blood glucose in food-deprived rats (NP, 83.2 +/- 2.1 mg/dL; HP, 91.2 +/- 2.1 mg/dL; P = 0.046) and increased plasma insulin in fed pups receiving the postweaning NP diet (gestation diet x postweaning diet, P = 0.02). Conclusion: Increasing the protein concentration of the rat dims' diet during gestation, and to a lesser extent during lactation, and of the pups' diet after weaning influenced pup phenotype, including body weight, fat accumulation, food intake, and glucose tolerance at 6 wk of age.