The aim of present study was to investigate the anti-obesity effect of Ilex paraguariensis extract and its molecular mechanism in rats rendered obese by a high-fat diet (HFD). I. paraguariensis extract supplementation significantly lowered body weight, visceral fat-pad weights, blood and hepatic lipid, glucose, insulin, and leptin levels of rats administered HFD. Feeding I. paraguariensis extract reversed the HFD-induced downregulation of the epididymal adipose tissue genes implicated in adipogenesis or thermogenesis, such as peroxisome proliferators' activated receptor gamma2, adipocyte fatty acid binding protein, sterol-regulatory-element-binding protein-1c, fatty acid synthase, HMG-CoA reductase, uncoupling protein 2, and uncoupling protein 3. Dietary supplementation with I. paraguariensis extract protected rats from the HFD-induced decreases in the phospho-AMP-activated protein kinase (AMPK)/AMPK and phospho-acetyl-CoA carboxylase (ACC)/ACC protein ratio related to fatty acid oxidation in the edipidymal adipose tissue. The present study reports that the I. paraguariensis extract can have a protective effect against a HFD-induced obesity in rats through an enhanced expression of uncoupling proteins and elevated AMPK phosphorylation in the visceral adipose tissue.