2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the β-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and β-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of β-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of β-catenin, as the 2DG stimulated accumulation of β-catenin, which is characterized by translocation of β-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of β-catenin degradation by inhibition of glycogen synthase kinase 3-β with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of β-catenin whereas the transcriptional level of β-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via β-catenin pathway in rabbit articular chondrocytes.