Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to form a variety of mesenchymal tissue types, which are a source of cells for bone tissue engineering applications. The present study attempted to establish an effective and convenient method for culturing BMSCs. Total bone marrow cells, which were harvested from rat femurs, were cultured and BMSCs were selected and expanded through passaging in vitro. Furthermore, the biological properties of BMSCs were investigated, specific surface antigen expression was assessed using flow cytometry and the multipotent differentiation potential characteristics were demonstrated using standard in vitro conditions. Monoptychial heterogeneous cells were obtained. A total of 98.4% of cells at passage 3 expressed cluster of differentiation (CD)29 and CD90, but not CD45. The cells were able to differentiate into osteogenic and adipogenic cells. In conclusion, BMSCs that are isolated from the rat bone marrow and exhibit the identified characteristics may be used as seed cells in bone tissue engineering.