2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), one of the flavonoids isolated and purified from the dried flower buds of Cleistocalyx operculatus, was explored for its function in glucose uptake/glycogen synthesis in insulin-sensitive tissue cells and its effect and mechanism on 3T3-L1 preadipocyte differentiation. DMC (10 μM) treatment remarkably promoted glucose uptake in differentiated 3T3-L1 adipocytes (P < 0.05 vs control group), whereas the glucose uptake in L6 myoblasts and glycogen synthesis in HepG2 hepatocytes were not affected by the treatment. DMC had paradoxical effects on lipid accumulation in 3T3-L1 cells compared with differentiation control. High concentrations of DMC (10 and 20 μM) markedly diminished lipid accumulation; however, a low concentration of DMC (2.5 μM) enhanced lipid storage in 3T3-L1 cells (P < 0.01 vs differentiation control group), and 5 μM DMC did not impose a significant effect. It was demonstrated that the effect of DMC in lipid accumulation was controlled by the expression of PPAR-γ.