To examine anti-adipogenic activity of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) isolated from the roots of Rubia cordifolia L., its effects on cell viability, apoptosis, and adipogenesis in 3T3-L1 preadipocytes were investigated. The inhibitory effect of CMEP-NQ on cell viability was more significant in differentiated mature adipocytes than in 3T3-L1 preadipocytes. In 3T3-L1 cells, the cytotoxicity of CMEP-NQ (20-40 μM) was accompanied by apoptotic events such as mitochondrial membrane potential loss, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and terminal deoxynucleotidyl transferase-meidated dUTP nick-end labeling-positive apoptotic DNA fragmentation. Although the presence of 10 μM CMEP-NQ during induced adipocytic differentiation of 3T3-L1 cells for 6 days failed to influence the cell viability, it did reduce the differentiation-associated accumulation of intracellular lipid by approximately 48.5%. A similar level of inhibition was observed when 10 μM CMEP-NQ was present during the early stage (Days 0-2) of the differentiation period. At the same time, the expressions of CCAAT/enhancer binding protein-α, peroxisome proliferator-activated receptor γ1, peroxisome proliferator-activated receptor γ2, and adiponectin were down-regulated. However, the presence of 10 μM CMEP-NQ during either the middle (Days 2-4) or late (Days 4-6) stage of the differentiation period caused the inhibition to a lesser extent. These results indicated that CMEP-NQ at high concentrations (20-40 μM) exerted cytotoxicity via inducing apoptosis, whereas CMEP-NQ at a low concentration (10 μM) suppressed adipocytic differentiation without exerting cytotoxicity in 3T3-L1 preadipocytes.