To investigate the effect of elevated plasma free fatty acid (FFA) concentrations on splanchnic glucose uptake (SGU), we measured SGU in nine healthy subjects (age, 44 ± 4 yr; body mass index, 27.4 ± 1.2 kg/m²; fasting plasma glucose, 5.2 ± 0.1 mmol/l) during an Intralipid-heparin (LIP) infusion and during a saline (Sal) infusion. SGU was estimated by the oral glucose load (OGL)-insulin clamp method: subjects received a 7-h euglycemic insulin (100 mU · m² · min¹) clamp, and a 75-g OGL was ingested 3 h after the insulin clamp was started. After glucose ingestion, the steady-state glucose infusion rate (GIR) during the insulin clamp was decreased to maintain euglycemia. SGU was calculated by subtracting the integrated decrease in GIR during the period after glucose ingestion from the ingested glucose load. [3-³H]glucose was infused during the initial 3 h of the insulin clamp to determine rates of endogenous glucose production (EGP) and glucose disappearance (R_d). During the 3-h euglycemic insulin clamp before glucose ingestion, R_d was decreased (8.8 ± 0.5 vs. 7.6 ± 0.5 mg · kg¹ · min¹, P < 0.01), and suppression of EGP was impaired (0.2 ± 0.04 vs. 0.07 ± 0.03 mg · kg¹ · min¹, P < 0.01). During the 4-h period after glucose ingestion, SGU was significantly increased during the LIP vs. Sal infusion study (30 ± 2 vs. 20 ± 2%, P < 0.005). In conclusion, an elevation in plasma FFA concentration impairs whole body glucose R_d and insulin-mediated suppression of EGP in healthy subjects but augments SGU.