Adiponectin is an adipokine that has pleiotropic beneficial roles in systemic insulin resistance and inflammation. Several recent clinical studies suggest that low serum levels of adiponectin are associated with increased risks of breast cancer. Here, we investigated the direct effects of adiponectin on breast cancer development in vitro and in vivo. Our results showed that adiponectin significantly attenuated the proliferations of two typical human breast cancer cells, MDA-MB-231 and T47D, in a cell type-specific manner. Further analysis revealed that adiponectin could induce apoptosis and arrest the cell cycle progression at G(0)-G(1) phase in MDA-MB-231 cells. Prolonged treatment with adiponectin in this cell line blocked serum-induced phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta), suppressed intracellular accumulation of beta-catenin and its nuclear activities, and consequently reduced expression of cyclin D1. Adiponectin-mediated suppression of cyclin D1 expression and attenuation of cell proliferation was abrogated by the GSK-3beta inhibitor lithium chloride. These results suggest that the inhibitory role of adiponectin on MDA-MB-231 cell growth might be attributed to its suppressive effects on the GSK-3beta/beta-catenin signaling pathway. Furthermore, our in vivo study showed that both supplementation of recombinant adiponectin and adenovirus-mediated overexpression of this adipokine substantially reduced the mammary tumorigenesis of MDA-MB-231 cells in female nude mice. Taken together, these data support the role of adiponectin as a negative regulator of breast cancer development and also suggest that adiponectin might represent a novel therapeutic target for this disease.