Adipocyte differentiation plays a pivotal role in the progression of obesity which is a major risk factor for several diseases such as diabetes, hypertension and coronary heart disease. In this study, the inhibitory effect of rhamnetin, a flavonoid compound, on adipogenesis in 3T3-L1 cells was investigated. Rhamnetin decreased the accumulation of lipid droplets, and inhibited the elevation of triglyceride content in the adipocytes (IC(50) = 17.3 μM). The expressions of PPARγ, C/EBPα, and perilipin, adipocyte differentiation markers, were significantly reduced by rhamnetin. Triglyceride biosynthesis and clonal expansion of adipocytes were completely inhibited during the early stage by rhamnetin. Additionally, rhamnetin significantly decreased the expression of C/EBPβ, an early stage marker. Our results indicate that suppression of clonal expansion during the early stage of adipogenesis by rhamnetin may be associated with inhibition of the C/EBPβ, C/EBPα, and PPARγ pathways.