Serum visfatin is elevated in insulin-resistant states, ie, obesity, type 2 diabetes mellitus, and polycystic ovary syndrome, thus linking visfatin with the pathogenesis of insulin resistance.Our objective was to evaluate the effect of hyperinsulinemia and the acute elevation of free fatty acids (FFAs) on serum visfatin in humans.We estimated serum visfatin during hyperinsulinemia and the insulin-resistant conditions caused by an acute elevation of FFAs in 19 healthy male volunteers (mean age, 25 ± 7 years; body mass index, 26 ± 4 kg/m(2)) at a university hospital.Intervention included a 6-hour euglycemic-hyperinsulinemic clamp without and with Intralipid/heparin infusion.Measurements were made of serum visfatin at baseline and at 2 and 6 hours during both clamp studies.Hyperinsulinemia resulted in a significant decrease in serum visfatin concentration (P = .043). Concomitant Intralipid/heparin infusion, which caused a reduction of insulin sensitivity by 40% (P < .0001), resulted in a marked increase in serum visfatin (P < .0001), which was already observed after 2 hours of FFAs increase (P < .0001). Serum visfatin during the clamp study after 2 and 6 hours of Intralipid/heparin infusion was significantly higher than respective values during the clamp study without the elevation of FFAs (both P < .0001). The increase in serum visfatin during Intralipid/heparin infusion was positively related to body weight (r = 0.54, P = .016) and γ-glutamyltransferase activity (r = 0.56, P = .011).Serum visfatin is differentially regulated by insulin and FFAs. One might hypothesize that the induction of insulin resistance by FFAs suppresses insulin inhibition of visfatin secretion, resulting in a serum visfatin increase in insulin-resistant conditions.