It has been well established that alendronate sodium-cholecalciferol(ASC) inhibits osteoclastic activity, and that this underlies its effectiveness as a treatment for metabolic bone diseases. Moreover, recent studies have indicated that ASC may have other effects on osteoblast activity. In this study, we evaluated the effect of ASC on the osteogenic differentiation of mouse mesenchymal stem cells. Accordingly, D1 cells(multipotent mouse mesenchymal stem cells) were cultured in osteogenic differentiation medium(ODM) for 3 days, and then treated with ASC for 2 days. The cell proliferation was determined using MTT assays, mineralization was followed by staining with alizarin red, alkaline phosphatase activity was measured using a commercial ELISA kit, calcification by energy dispersive X-ray spectrophotometry(EDX), osteogenic gene expression using RT-PCR, and changes in CD 44 expression by confocal microscopy and FACS. D1 cells differentiated into osteoblasts in the presence of ODM, as confirmed by positive Alizarin red S staining, increased ALP activity and osteocalcin mRNA expression, a calcium peak by EDX, and by positive immunofluorescence staining against CD 44(an antigen present on osteoblasts). After ASC had been added to ODM, osteogenic differentiation was enhanced, as confirmed by Alizarin red S staining, elevated ALP activity and osteocalcin mRNA expression, a greater calcium peak by EDX, and by immunofluorescence staining against CD 44 by FACS. This study demonstrates that ASC enhances osteogenic differentiation when treated to mouse mesenchymal stem cells in ODM. The authors suggest that ASC increases bone density by increasing the osteogenic differentiation of mesenchymal stem cells and by inhibiting osteoclast activity.