Obesity and its global prevalence has become a threat to human health, while its pharmacotherapy via the application of natural products is still underdeveloped. Here, we probed how 4,5,4'-trihydroxy-3,3'-dimethoxybibenzyl (TDB) derived from an orchid (Dendrobium ellipsophyllum) could exert its roles on the differentiation and function of murine (3T3-L1) and human (PCS-210-010) pre-adipocytes and offer some implications to modulate obesity. Cytotoxic effects of TDB on adipocytes were 2-fold lower than those detected with pre-adipocytes, and no significant difference was detected in cytotoxic profiles between both cell lineages. TDB in a dose-dependent manner decreased cellular lipid accumulation and enhanced lipolysis of both cell lines assessed at early differentiation and during maturation. Underlining molecular mechanisms proved that TBD paused the cell cycle progression by regulating inducers and inhibitors in mitotic clonal expansion, leading to growth arrest of pre-adipocytes at the G0/G1 phase. The compound also governed adipocyte differentiation by repressing expressions of crucial adipogenic regulators and effectors through deactivating the AKT/GSK-3β signaling pathway and activating the AMPK-ACC pathway. To this end, TDB has shown its pharmaceutical potential for modulating adipocyte development and function, and it would be a promising candidate for further assessments as a therapeutic agent to defeat obesity.