Glutamine (Gln)-supplemented total parenteral nutrition (TPN) has been shown to improve mucosal adaptation after massive small bowel resection (SBR); however, its influences on intestinal amino acid metabolism remain unknown. In this study, intestinal amino acid flux, circulating plasma aminogram, mucosal glutaminase activity and protein, and DNA content were measured 7 days after massive SBR in rats receiving either standard (Std) or Gln-supplemented TPN. Sham-operated rats and rats fed chow after enterectomy served as controls. The uptake of Gln and the release of citrulline (Cit) by the remaining intestine was significantly decreased, with reduced mucosal glutaminase activity after SBR in the Chow and Std-TPN groups. Glutamine supplementation resulted in significantly increased gut Gln uptake compared with Std-TPN (P < 0.01). Mucosal glutaminase activity, mucosal protein, and DNA content was also increased by Gln; however, the gut release of Cit remained unchanged (P > 0.05). The subsequent decrease in circulating arginine (Arg) in the Gln-TPN group compared with the Std-TPN group (P < 0.05) was attributed to an insufficient exogenous supply. These findings show that Gln-supplemented TPN improves mucosal growth and gut Gln uptake after SBR. However, the intestinal production of Cit, which remained low in both TPN groups, may lead to an insufficiency of endogenous Arg synthesis. Thus, both Gln and Arg may be essential amino acids after SBR.