In this study, we investigate the ability of reversine to stimulate adipocyte differentiation and its effect on cellular signaling pathways associated with adipocyte differentiation. Our data show that reversine treatment of 3T3-L1 cells under differentiation conditions synergistically enhances adipocyte differentiation and the expression of adipogenic marker genes such as aP2, PPAR-gamma, resistin, C/EBPalpha, and adiponectin. In parallel, reversine treatment leads to a selective downregulation of Akt and p70(s6k) signaling pathways, but not the ERK pathway. Furthermore, reversine stimulation of adipocyte differentiation seems to be quite different from troglitazone's action, because reversine treatment does not induce the transcriptional activation of PPAR-gamma and troglitazone does not affect the Akt and p70(s6k) signaling pathways. Taken together, our data clearly demonstrate the ability of reversine to stimulate adipocyte differentiation, which is independent of the Akt and p70(s6k) signaling pathways.