Ectopic fat storage was caused by metabolic disorder and resulted in metabolic diseases such as nonalcoholic fatty liver disease (NAFLD), obesity and hyperlipidemia. Fat burning was considered as an effective way for ameliorating metabolic diseases. Herein, fat mobilization failed to improve fat consumption effectively, promoting fatty acid oxidation, thus was consider as the key step of fat burning process. After searching for the natural activators, we found that theacrine, obtained from Camellia assamica var. Kucha, a special tea plant in Yunnan province of China, improves palmitate oxidation and induces a subsequent inhibition of fatty acid elongation by using fatty acid flux analysis. In addition, theacrine reduced body fat by increasing the mid-dorsal temperature in obese mice and focal uptake of 18F-FDG in brown adipose tissues (BAT). Moreover, theacrine ameliorated triglyceride metabolic disorder in restrained mice through increasing lipoprotein lipase activity, thereby promoting fatty acid metabolism. Our findings also demonstrated theacrine protected against diet-induced or stress-susceptible NAFLD. As acylcarnitine metabolism disorder contributed a lot to the development of NAFLD, the reductions of long-chain or short-chain acylcarnitines and the increased ratios of longer/shorter-chain acylcarnitines by theacrine could explain its improvement involved in fatty acid oxidation. In molecular mechanism, theacrine increased the activity of hepatic long-chain a cyl coenzyme A dehydrogenase ( LCAD ) through deacetylation, and the deacetylation involved theacrine’s activation of the mitochondrial deacetylase Sirtuin 3 (SIRT3). On the basis of theacrine’s promoting fatty acid oxidation and energy expenditure, theacrine ameliorated acylcarnitine metabolism disorder in diet-induced or stress-susceptible NAFLD through the SIRT3/LCAD signaling pathway, thereby promoting fat burning in liver and adipose tissues. SIRT3 is confirmed as a target involved theacrine’s fat burning effect. Finally, it will be worth to develop a theacrine-containing medication to prevent ectopic fat storage in metabolic diseases like NAFLD, obesity and hyperlipidemia.