alpha- and beta-Catenin link cadherins to the actin-based cytoskeleton at adherens junctions and regulate cell-cell adhesion. Although roles of cadherins and canonical Wnt-/beta-catenin-signaling in osteoblastic differentiation have been extensively studied, the role of alpha-catenin is not known. Murine embryonic mesenchymal stem cells, C3H10T1/2 cells, were transduced with retrovirus encoding alpha-catenin (MSCV-alpha-catenin-HA-GFP). In the presence of Wnt-3A conditioned medium or osteogenic medium (beta-glycerol phosphate and ascorbic acid), cells overexpressing alpha-catenin showed enhanced osteoblastic differentiation as measured by alkaline phosphatase (ALP) staining and ALP activity assay compared to cells transduced with empty virus (MSCV-GFP). In addition, mRNA expression of osteocalcin and Runx2 was significantly increased compared to control. Cell aggregation assay revealed that alpha-catenin overexpression has significantly increased cell-cell aggregation. However, cellular beta-catenin levels (total, cytoplasmic-nuclear ratio) and beta-catenin-TCF/LEF transcriptional activity did not change by overexpression of alpha-catenin. Knock-down of alpha-catenin using siRNA decreased osteoblastic differentiation as measured by ALP assay. These results suggest that alpha-catenin overexpression increases osteoblastic differentiation by increasing cell-cell adhesion rather than Wnt-/beta-catenin-signaling.