Objective: To further investigate the osteogenic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by density gradient centrifugation method and amplified in the flasks, using the osteogenic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone-seeking fluorescence (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue-Sirius red (AS) staining, and scanning electron microscope. Results: After being passaged, the marrow stromal stem cells increased in number, became confluent and formed multi-layer structure. The stromal stem cells excreted innumerable tiny granules, heaping up on the cell body and merging gradually into foggy substances. These foggy substances kept on enlarging and formed round, oval, or flake-like nodules. These nodules revealed bright golden yellow fluorescence under fluorescence microscope when labelled with tetracycline. Histochemical study with specific new bone staining with ARS revealed positive calcium reaction, both denoting that they were newly formed bone tissues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different configurations were found. They were globular cells, spindle-shaped cells and polygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle-shaped and irregularly rectangular crystals also appeared and agglomerated with the granules to form nodules and trabecula-like or flake-like structures.Conclusions: Sequence of events of bone formation by rabbit marrow stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast differentiation from marrow stromal stem cells and the possible application in orthopaedics.