In order to determine anti-adipogenic effect, this study investigated 1β-hydroxy-2-oxopomolic acid (HOA) isolated from Agrimonia pilosa inhibits adipocyte differentiation and expression of adipogenic marker genes, such as peroxisome proliferator activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), glucose transporter 4 (GLUT4), adiponectin, adipocyte fatty acid-binding protein 2 (aP2), adipocyte determination and differentiation factor 1/sterol regulatory element binding protein 1c (ADD1/SREBP1c), resistin, and fatty acid synthase (Fas) in 3T3-L1 preadipocyte. We demonstrated that HOA induced a significant decrease in lipid accumulation and expression of adipogenic marker genes in a dose-dependent manner. In addition, HOA reduced the transcripitional activity of PPARγ induced by troglitazone, a potent diabetes agent; it also suppressed expression of PPARγ and C/EBPα protein levels. Our data suggest that HOA isolated from Agrimonia pilosa inhibits adipocyte differentiation through downregulation of various adipocytokines by blocking PPARγ and C/EBPα expression.