OBJECTIVE - Lipocalin (LCN) 2 belongs to the lipocalin subfamily of low-molecular mass-secreted proteins that bind small hydrophobic molecules. LCN2 has been recently characterized as an adipose-derived cytokine, and its expression is upregulated in adipose tissue in genetically obese rodents. The objective of this study was to investigate the role of LCN2 in diet-induced insulin resistance and metabolic homeostasis in vivo. RESEARCH DESIGN AND METHODS - Systemic insulin sensitivity, adaptive thermogenesis, and serum metabolic and lipid profile were assessed in LCN2-deficient mice fed a high-fat diet (HFD) or regular chow diet. RESULTS - The molecular disruption of LCN2 in mice resulted in significantly potentiated diet-induced obesity, dyslipidemia, fatty liver disease, and insulin resistance. LCN2-/- mice exhibit impaired adaptive thermogenesis and cold intolerance. Gene expression patterns in white and brown adipose tissue, liver, and muscle indicate that LCN2 -/- mice have increased hepatic gluconeogenesis, decreased mitochondrial oxidative capacity, impaired lipid metabolism, and increased inflammatory state under the HFD condition. CONCLUSIONS - LCN2 has a novel role in adaptive thermoregulation and diet-induced insulin resistance.