Background and Purpose: Pharmacological intervention to induce browning of white adipose tissue provides a promising anti-obesity therapy. The fruits of Garcinia cambogia (Clusiaceae) have been widely applied to manage body weight; however, the chemical principles remain unclear. The current study aims to discover browning inducers from the fruits of G. cambogia and investigate the underlying mechanisms. Experimental Approach: The bioactivity-based molecular networking and Oil Red O staining on 3T3-L1 and C3H10T1/2 adipocytes were applied for guided isolation. High-fat diet-induced obese mice were recruited to evaluate the anti-obesity activity. Key Results: The bioactivity-based molecular networking-guided isolation yielded several polycyclic polyprenylated acylphloroglucinols from the fruits of G. cambogia with lipid-lowering effect in adipocytes, including guttiferone J (GOJ), garcinol and 14-deoxygarcinol. As the most potent one, GOJ (10 μM) reduced lipid accumulation by 70% and 76% in 3T3-L1 and C3H10T1/2 adipocytes, respectively. Furthermore, GOJ (2.5–10 μM) increased the expression of the deacetylase sirtuin 3 (SIRT3) and activated it, which, in turn, reduced the acetylation level of PPARγ coactivator-1α to boost mitochondrial biogenesis and promoted uncoupling protein 1 expression to enhance thermogenesis, resulting in browning of adipocytes. In high-fat diet-induced-obese mice, GOJ (10 and 20 mg·kg·day for 12 weeks) protected against adiposity, hyperlipidaemia, insulin resistance and liver lipotoxicity, through boosting SIRT3-mediated browning of inguinal adipose tissue. Conclusion and Implications: GOJ represents a new scaffold of thermogenic inducer, which is responsible for the anti-obesity property of G. cambogia and can be further developed as a candidate for treating obesity and its related disorders.