Natural products are known to be a rich source of potential compounds for metabolic diseases. In this study, we aim to identify the lead compounds with anti-adipogenic and anti-obesity effects in Cimicifuga foetida L. (CF). Five high-content chemicals from ethyl acetate extract of CF (ECF) were isolated to investigate their effects on adipogenesis in 3T3-L1 preadipocytes. Actein is identified to possess substantial anti-adipogenic activity in 3T3-L1 adipocytes, which exerts its anti-adipogenic effect throughout the entire adipocyte differentiation stages. Dietary actein ameliorates diet-induced obesity, improves the disturbed serum lipid levels, and enhances white fat browning in diet-induced obese mice. Metabolomic analysis using urine and serum samples from mice revealed that actein treatment reverses the dysregulation of γ-linolenic acid and lecithin in obese mice, thus ameliorating the disturbance of linoleic acid metabolism induced by high-fat diet, suggesting that actein can be used as a lead compound with the potential to develop new therapies for obesity and related metabolic disorders.