Context The detrimental effect of elevated free fatty acids (FFA) on insulin sensitivity can be improved by thiazolidinediones (TZD) in patients with type 2 diabetes mellitus. It is unknown if this salutary action of TZD is associated with altered release of the insulin-mimetic adipocytokine visfatin. Objectives. In this study we have investigated whether visfatin concentrations are altered by FFA and TZD treatment. Design and Patients. In a randomized, double-blind, placebo-controlled, parallel-group study 16 healthy volunteers received an infusion of triglycerides/heparin to increase plasma FFA after three weeks of treatment with rosiglitazone (8 mg/d, n=8) or placebo (n=8) and circulating plasma visfatin was measured. As a corollary, human adipocytes were incubated with synthetic fatty acids and rosiglitazone to assess visfatin release in vitro. Results. Rosiglitazone treatment increased systemic plasma visfatin concentrations from 0.6±0.1 to 1.7±0.2 ng/ml (p<0.01). Lipid infusion caused a marked elevation of plasma FFA but had no effect on circulating visfatin in controls. In contrast, elevated visfatin concentrations in subjects receiving rosiglitazone were normalized by lipid infusion. In isolated adipocytes, visfatin was released into supernatant media by acute addition and long term treatment of rosiglitazone. This secretion was blocked by synthetic fatty acids and by inhibition of phosphadityl-inositol-(PI)3-kinase or AKT. Conclusion. Release of the insulin-mimetic visfatin may represent a major mechanism of metabolic thiazolidinedione action. The presence of FFA antagonizes this action, which may have implications for visfatin bioactivity.