This study investigated the anti-obesity effects of the ethanol extract of Cimicifuga heracleifolia Kom. (CHEE) and its molecular mechanisms using 3T3-L1 preadipocytes. The ethanol extract of Cimicifuga heracleifolia Kom. was prepared by extraction with 70% ethanol. 3T3-L1 preadipocytes were treated with CHEE, and the levels of lipid accumulation, protein expression of adipogenic transcription factors, and AMP-activated protein kinase (AMPK) phosphorylation were measured. Lipid accumulationdetermined by Oil Red O staining, western blotting, and real-time PCR. The Chemical components of CHEE were analyzed by ultra-performance liquid chromatography coupled with a diode array detector (UPLC-DAD). CHEE inhibited lipid accumulation in 3T3-L1 cells in a dose-dependent manner without showing cytotoxicity up to 200 μg/mL with approximately 45% reduction at 200 μg/mL (p<0.01). CHEE significantly decreased the protein and mRNA expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), CCAAT/enhancer-binding protein beta (C/EBPβ), and CCAAT/enhancer-binding protein delta (C/EBPδ). Additionally, CHEE treatment significantly increased phospho-AMPK levels, and UPLC analysis revealed that CHEE contained 18.25 mg/g of ferulic acid. This study demonstrated that CHEE suppresses 3T3-L1 preadipocyte differentiation by inhibiting the expression of PPARγ and C/EBPα through AMPK activation. These results suggest that the ethanol extract of Cimicifuga heracleifolia Kom. has potential for development as a therapeutic agent for obesity and metabolic diseases, mediated through AMPK-related signaling pathways. The anti-obesity activity of CHEE appears to result from multiple bioactive components working synergistically. Further study using AMPK inhibitors and component-specific analysis will be needed to elucidate the precise mechanism of action.