In enterocytes, glutamine serves as the major source of energy; another metabolic fate of glutamine is conversion to citrulline. Because sepsis can affect gut function and integrity, alterations in glutamine metabolism may exist and lead to decreased citrulline production. This study aimed to investigate how sepsis affects glutamine metabolism, including its conversion to citrulline, by measuring glutamine and citrulline flux, fractional splanchnic extraction of glutamine and leucine, and the contribution of glutamine nitrogen to citrulline in septic patients and healthy controls. Eight patients with severe sepsis and 10 healthy controls were given primed, constant intravenous infusion of ²H₂-citrulline and sequential administration of intravenous and enteral α-¹⁵N-glutamine and ¹³C-leucine in the postabsorptive state. The results showed that compared with healthy controls, septic patients had a significantly lower whole-body citrulline flux and plasma concentration, higher endogenous leucine flux, and higher glutamine clearance. Fractional splanchnic extraction of leucine was higher in septic patients compared with controls, but fractional extraction of glutamine was not different. The majority of the ¹⁵N label transferred from glutamine to citrulline was found at the alpha position. These results demonstrate that lower glutamine plasma concentrations in sepsis were a result of increased glutamine clearance. Despite adequate splanchnic uptake of glutamine, there is decreased production of citrulline, suggesting a defect in the metabolic conversion of glutamine to citrulline, decreased uptake of glutamine by the enterocyte but increased uptake by the liver, and/or shunting of glutamine to other metabolic pathways.