Activation of inflammatory pathways is known to accompany development of obesity-induced nonalcoholic fatty liver disease (NAFLD), insulin resistance and type 2 diabetes. In addition to caspase-1, the neutrophil serine proteases proteinase 3, neutrophil elastase and cathepsin G are able to process the inactive proinflammatory mediators interleukin (IL)-1β and IL-18 to their bioactive forms, thereby regulating inflammatory responses. In this study, we investigated whether proteinase 3 is involved in obesity-induced development of insulin resistance and NAFLD. We investigated the development of NAFLD and insulin resistance in mice deficient for neutrophil elastase/proteinase 3 and neutrophil elastase/cathepsin G and in wild-type mice treated with the neutrophil serine proteinase inhibitor human α-1 antitrypsin. Expression profiling of metabolically relevant tissues obtained from insulin-resistant mice showed that expression of proteinase 3 was specifically upregulated in the liver, whereas neutrophil elastase, cathepsin G and caspase-1 were not. Neutrophil elastase/proteinase 3-deficient mice showed strongly reduced levels of lipids in the liver after being fed a high-fat diet. Moreover, these mice were resistant to high–fat–diet-induced weight gain, inflammation and insulin resistance. Injection of proteinase 3 exacerbated insulin resistance in caspase-1–/– mice, indicating that proteinase 3 acts independently of caspase-1. Treatment with α-1 antitrypsin during the last 10 d of a 16-wk high-fat diet reduced hepatic lipid content and decreased fasting glucose levels. We conclude that proteinase 3 is involved in NAFLD and insulin resistance and that inhibition of proteinase 3 may have therapeutic potential.