Background: Dietary protein is required to attenuate the loss of muscle mass and to support recovery during a period of hospitalization. Jejunal feeding is preferred over gastric feeding in patients who are intolerant of gastric feeding. However, the impact of gastric vs. jejunal feeding on postprandial dietary protein digestion and absorption kinetics in vivo in humans remains largely unexplored. Objective: We compared the impact of gastric vs. jejunal feeding on subsequent dietary protein digestion and amino acid (AA) absorption in vivo in healthy young men. Methods: In a randomized crossover study design, 11 healthy young men (aged 21 ± 2 y) were administered 25 g specifically produced intrinsically l-[1-13C]phenylalanine–labeled intact casein via a nasogastric and a nasojejunal tube placed ~30 cm distal to the ligament of Treitz. Protein was provided in a 240-mL solution administered over a 65-min period in both feeding regimens. Blood samples were collected during the 7-h postprandial period to assess the increase in plasma AA concentrations and dietary protein–derived plasma l-[1-13C]phenylalanine enrichment. Results: Jejunal feeding compared with gastric feeding resulted in higher peak plasma phenylalanine, leucine, total essential AA (EAA), and total AA concentrations (all P <0.05). This was attributed to a more rapid release of dietary protein–derived AAs into the circulation, as evidenced by a higher peak plasma l-[1-13C]phenylalanine enrichment concentration (2.9 ± 0.2 vs. 2.2 ± 0.2 mole percent excess; P <0.05). The total postprandial plasma AA incremental area under the curve and time to peak did not differ after jejunal vs. gastric feeding. Plasma insulin concentrations increased to a greater extent after jejunal feeding when compared with gastric feeding (275 ± 38 vs. 178 ± 38 pmol/L; P <0.05). Conclusions: Jejunal feeding of intact casein is followed by more rapid protein digestion and AA absorption when compared with gastric feeding in healthy young men. The greater postprandial increase in circulating EAA concentrations may allow a more robust increase in muscle protein synthesis rate after jejunal vs. gastric casein feeding. This trial was registered at trialregister.nl as NTR2801.