Adipose-derived stem cells (ASCs) are considered to be multipotent mesenchymal stem cells that are easily induced to differentiate into functional osteoblasts both in vitro and in vivo. Osterix (Osx) is a zinc finger-containing transcription factor of Sp gene family, which plays important roles in bone development and mineralization. In this study, we hypothesized that overexpression of Osx in murine ASCs would promote their osteogenic differentiation in vitro. A plasmid expressing Osx (pcDNA3.1-Osx) was constructed and applied to transfect monolayers of murine ASCs. Then expression of bone-related genes, nodule formation, proliferation rate, and alkaline phosphatase activity were examined to evaluate the osteogenic potential of ASCs with pcDNA3.1-Osx transfection. Results of RT-PCR and immunohistochemistry showed that pcDNA3.1-Osx transfection enhanced the expression of bone matrix proteins, such as bone sialoprotein, osteocalcin, osteopontin, and Collagen type I in ASCs. At the same time, overexpression of Osx in ASCs enhanced alkaline phosphatase activity and capability to form mineralized nodules, while not inhibited their proliferation rate. These results indicated that pcDNA3.1-Osx transfection promoted the osteogenic differentiation of ASCs, while not affecting their proliferative ability. Since they can be easily isolated and genetically modified, ASCs are hopeful cell sources in the further application of hard tissue engineering.