The prevalence of obesity has increased dramatically worldwide in the past ~50 years. Searching for safe and effective anti-obesity strategies are urgently needed. Lactucin, a plant-derived natural small molecule, is known for anti-malaria and anti-hyperalgesia. The study is to investigate whether lactucin plays a key role in adipogenesis. To this end, in vivo male C57BL/6 mice fed a high-fat diet (HFD) were treated with 20 mg/kg/day of lactucin or vehicle by gavage for seven weeks. Compared with vehicle-treated controls, Lactucin-treated mice showed lower body mass and mass of adipose tissue. Consistently, in vitro 3T3-L1 cells were treated with 20 μM of lactucin. Compared to controls, lactucin-treated cells showed significantly less lipid accumulation during adipocyte differentiation and lower levels of lipid synthesis markers. Mechanistically, we showed the anti-adipogenic property of lactucin was largely limited to the early stage of adipogenesis. Lactucin-treated cells fail to undergo mitotic clonal expansion (MCE). Further studies demonstrate that lactucin-induced MCE arrests might result from reduced phosphorylation of JAK2 and STAT3. We then asked whether activation of JAK2/STAT3 would restore the inhibitory effect of lactucin on adipogenesis with pharmacological STAT3 activator colivelin. Our results revealed similar levels of lipid accumulation between lactucin-treated cells and controls in the presence of colivelin, indicating that inactivation of STAT3 is the limiting factor for the anti-adipogenesis of lactucin in these cells. Together, our results provide the indication that lactucin exerts an anti-adipogenesis effect, which may open new therapeutic options for obesity.