The objective was to examine the effectiveness of mate tea (MT, Ilex paraguariensis St. Hilaire) and caffeine from mate tea (MC) on in vitro lipid accumulation and in vivo diet-driven-obesity. MC and decaffeinated mate (DM) were obtained using supercritical CO2 extraction and mainly composed of caffeine and caffeoylquinic acids, respectively. MC reduced lipid accumulation (41%) via downregulation of fatty acid synthase (Fasn) (39%) in 3T3-L1 adipocytes. Rats fed a high-fat-high-sucrose-diet and 0.1% of caffeine from MC, MT, or DM. MC attenuated weight gain (16%) and body fat accumulation (22%). MC reduced Fasn expression in both adipose tissue (66%) and liver (37%). MC diminished pyruvate kinase (PK, 59%) and microsomal triglyceride transfer protein (MTP, 50%) hepatic expression. In silica, neochlorogenic acid interacted with PK and MTP allosteric sites. FAS beta-ketoacyl reductase domain showed the highest affinity to 3,5-dicaffeoylquinic acid. Caffeine suppressed lipid accumulation and body weight gain, through the modulation of lipogenic gene expression.