Potentilla longifolia is a kind of Chaoyao medicine, which is a branch of traditional Chinese medicine. The plant is often referred to as ganyancao or ganyearmcao, which means that it has a significant therapeutic effect on liver inflammation. In previous experiments, we found that a water extract of ganyearmcao inhibited lipid accumulation. In the present study, we isolated one new (ganyearmcaoone A, 1) and eight known compounds (2-9) from a water extract of the dried roots of ganyearmcao; all of the compounds were isolated for the first time from this medicinal plant. We elucidated the chemical structures of these compounds using comprehensive analyses of HR-ESI-MS and 1D, 2D NMR. We evaluated the inhibitory effects of the nine compounds on lipid accumulation in 3T3-L1 cells; we did so using photographic and quantitative assessments of the lipid content with oil red O staining and by measuring triglyceride levels. Compared with the control, compounds 6 and 9 significantly inhibited differentiation of 3T3-L1 cells and lipid accumulation. Compound 1 showed potential inhibitory effects on lipid accumulation. Molecular docking results indicated that compounds 6 and 9 may efficiently bind to AMPK and its downstream kinase (SCD1), thereby inhibiting lipid accumulation. Our results demonstrate that ganyearmcao and its components may play an important role in treating diseases related to lipid accumulation in the future.