Obesity is a global health burden for which we do not yet have effective treatments for prevention or therapy. Plants are an invaluable source of bioactive leads possessing anti-adipogenic potential. Ethnopharmacological use of Ononis spinosa L. roots (OSR) for treatment of obesity and metabolic disorders requires а scientific rationale. The current study examined the anti-adipogenic capacity of OSR and its secondary metabolites ononin (ONON) and maackiain (MACK) in human adipocytes as an in vitro model of obesity. Both ONON and MACK diminished lipid accumulation during adipocyte differentiation. Molecular docking analysis exposed the potential interactions between MACK or ONON and target regulatory adipogenic proteins. Furthermore, results from an RT-qPCR analysis disclosed significant upregulation of AMPK by MACK and ONON treatment. In addition, ONON increased SIRT1, PI3K and ACC mRNA expression, while MACK notably downregulated CEBPA, AKT, SREBP1, ACC and ADIPOQ. The protein level of PI3K, C/EBPα, PPARγ and adiponectin was reduced upon MACK treatment in a concentration-dependent manner. Similarly, ONON suppressed PI3K, PPARγ and adiponectin protein abundance. Finally, our study provides evidence that ONON exerts anti-adipogenic effect by upregulation of SIRT1 and inhibition of PI3K, PPARγ and adiponectin, while MACK induced strong inhibitory effect on adipogenesis via hampering PI3K, PPARγ/C/EBPα signaling and anti-lipogenic effect through downregulation of SREBP1 and ACC. Even though OSR does not hamper adipogenic differentiation, it could be exploited as a source of natural leads with anti-adipogenic potential. The multidirectional mechanism of action of MACK warrant further validation in the context of in vivo obesity models.