OBJECTIVE: Lipocalin-2, a novel adipokine, has been shown to be elevated in obese, insulin-resistant, and diabetic subjects. We therefore sought to study the ex vivo and in vivo effects of insulin on lipocalin-2 levels in humans. RESEARCH DESIGN AND METHODS: We investigated the in vivo effects of insulin (hyperinsulinemia) on circulating lipocalin-2 levels by enzyme-linked immunosorbent assay via a prolonged insulin-glucose infusion. The ex vivo effect of insulin on adipose tissue lipocalin-2 protein production and secretion into conditioned media was assessed by Western blotting and enzyme-linked immunosorbent assay, respectively. RESULTS: Hyperinsulinemic induction in human subjects significantly increased circulating lipocalin-2 levels (P < 0.01). Also, in omental adipose tissue explants, insulin caused a significant dose-dependent increase in lipocalin-2 protein production and secretion into conditioned media (P < 0.05, P < 0.01, respectively); these effects were negated by both phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase inhibitors. CONCLUSIONS: Lipocalin-2 is upregulated by insulin via phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways.