Mesenchymal stem cells (MSCs), known as seed cells, have been shown to be useful in repairing damaged or diseased tissues and organs in the past. However, its clinical application in vivo is limited by individual differences and diversity of sources. In contrast, periosteum or periosteum-derived stem cells (PSCS) are more appropriate for fracture or cartilage damage. PC is also known as MSCs, but it is not clear whether PC is a class of cells of MSCs. Our objective was to isolate the PC of the periosteum of the duck embryo tibia and study its properties. The femoral periosteum of duck embryos at 12 days of age was easily collected, and the isolated cells showed typical stem cell morphology by enzyme digestion and adhesion method. Both immunofluorescence detection and RT-PCR showed that PSC could express CD90, CD29, CD166 and CD44 on the surface of MSCs, but not CD34 on the surface of hematopoietic stem cells. Under different culture conditions, PSC was proved to have the ability to differentiate into adipocytes, osteoblasts and chondroblasts in vitro. In addition, PC also has a strong ability to proliferate and self-renewal ability. In conclusion, our study suggests that PSCS may contain MSCs, which can be used as one of the candidate resources for clinical treatment of bone diseases.