Objective: Chronic excessive food intake leads to energy imbalance, resulting in hepatic steatosis and inflammation. Interleukin-32 (IL-32) is known to be a pro-inflammatory cytokine associated with chronic inflammation and cancer. Therefore, the relationship between IL-32 and chronic excessive food intakeinduced liver disease was investigated. Methods: Male IL-32b transgenic and wild-type mice were fed a high-fat diet (HFD) for 15 weeks. They were compared with wild-type mice on a standard chow diet. Daily food intake, body and liver weight, serum biochemistry, histopathological analysis of the liver, and hepatic immune response were determined. Results: IL-32b mice on HFD showed lower lipid accumulation, reduced infiltration of immune cells, and lower production of pro-inflammatory cytokines in the liver. The expression of the peroxisome proliferator-activated receptor c (PPARc) was downregulated and the adenosine 50-monophosphate (AMP)-activated protein kinase (AMPK) was activated in the liver of IL-32b mice compared to wild-type mice. Furthermore, IL-32b over-expression activated the AMPK pathway and IL-32b downregulation inactivated the AMPK pathway in HepG2 cells under high-glucose conditions. Conclusions: These data suggest that IL-32b modulates lipid accumulation through inhibition of PPARc expression and AMPK activation.