Background: Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea and has been considered a natural agent that can help to reduce the risk of obesity. Objective: The aim of this study was to investigate the effects of EGCG on thermogenesis and mitochondrial biogenesis in brown adipose tissue (BAT) of diet-induced obese mice. Methods: Male C57BL/6J mice were provided a high-fat diet for 8 weeks to induce obesity, following which they were divided into two groups: one on a high-fat control diet and the other on a 0.2% EGCG (w/w)-supplemented high-fat diet for another 8 weeks. Results: The EGCG-supplemented group showed decreased body weight gain, and plasma and liver lipids. EGCG-fed mice exhibited higher body temperature and mitochondrial DNA (mtDNA) content in BAT. The messenger RNA levels of genes related to thermogenesis and mitochondrial biogenesis in BAT were increased by EGCG. Moreover, adenosine monophosphate-activated protein kinase (AMPK) activity in BAT was stimulated by EGCG. Conclusions: The results suggest that EGCG may have anti-obesity properties through BAT thermogenesis and mitochondria biogenesis, which are partially associated with the regulation of genes related to thermogenesis and mitochondria biogenesis, and the increase in mtDNA replication and AMPK activation in BAT of diet-induced obese mice.