Liver-fatty-acid-binding-protein (L-FABP) is a highly conserved key factor in lipid metabolism. Amino-acid replacements in L-FABP might alter its function and thereby affect glucose metabolism in lipid-exposed subjects, as indicated by studies in L-FABP knockout mice. Amino-acid replacements in L-FABP were investigated in a cohort of 1453 Caucasian subjects. Endogenous glucose production (EGP), gluconeogenesis, and glycogenolysis were measured in healthy carriers of the only common Thr94-to-Ala amino acid replacement (Ala/Ala94) versus age, gender, and BMI matched wild-type (Thr/Thr-94) controls at baseline and after 320-min lipid/heparin-somatostatin-insulin-glucagon clamps (n = 18). Whole-body glucose disposal was further investigated (subset, n = 13), using euglycemic-hyperinsulinemic clamps without and with lipid/heparin infusion. In the entire cohort, the only common Ala/Ala-94 mutation was significantly associated with reduced body weight, which is in agreement with a previous report. In lipid-exposed, individually matched subjects, there was a genotype vs. lipid-treatment interaction for EGP (P = 0.009), mainly driven by reduced glycogenolysis in Ala/Ala94-carriers (0.46±0.05 vs. 0.59±0.05 mg·kg^-1·min^-1, P = 0.013). The lipid-induced elevation of plasma glucose levels was smaller in Ala/Ala94-carriers in comparison to wild-types (P< 0.0001). Whole-body glucose disposal was not different between lipid-exposed L-FABP genotypes. In summary the Ala/Ala94-mutation significantly contributed to reduced glycogenolysis and less severe hyperglycemia in lipid-exposed humans, and was further associated with reduced body weight in a large cohort. Data clearly show that investigation of L-FABP phenotypes in the basal, overnight fasted state yielded incomplete information and a challenge test was essential to detect phenotypical differences in glucose metabolism between L-FABP genotypes.