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It has been reported that H. mollendorffii roots (HMR) have various pharmacological activities such as anti‐inflammatory activity and immunostimulatory activity. However, the anti‐obesity activity of HMR has not been studied. Thus, we evaluated in vitro anti‐obesity of HMR in mouse preadipocytes, 3T3‐L1 cells. HMR reduced the lipid accumulation and triglyceride (TG) contents in 3T3‐L1 cells. HMR inhibited the protein expressions such as CCAAT/enhancer‐binding protein alpha (CEBPα), peroxisome proliferator‐activated receptor gamma (PPARγ), perilipin‐1, adiponectin, fatty acid‐binding protein 4 (FABP4), fatty acid synthase (FAS), and acetyl‐CoA carboxylase (ACC) related to the lipid accumulation of the mature adipocytes. In addition, HMR induced the proteasomal degradation of CEBPα related to the differentiation of the preadipocytes into the mature adipocytes by activating c‐Jun N‐terminal kinases (JNK) and glycogen synthase kinase 3 beta (GSK3β). Based on the results of this study, HMR inhibited the differentiation of preadipocytes into mature adipocytes through the CEBPα degradation via JNK and GSK3β activation and subsequently blocked lipid accumulation of mature adipocytes through inhibiting lipid accumulation‐related proteins such as CEBPα, PPARγ, perilipin‐1, adiponectin, FABP4, FAS, and ACC. [ABSTRACT FROM AUTHOR]