Bone tissue engineering is a promising cell-based strategy to treat bone defects. Mesenchymal stem cells from adult human bone marrow (hBMSCs) are a frequently used cellular source for bone tissue generation. However, the low frequency of these stem cells in adult bone marrow and their limited proliferation restrict their clinical utility. An alternative source of MSCs is the periosteum-derived cells, and these cells appear to be easy to harvest and expand ex vivo. We isolated human metaphyseal periosteum-derived cells (hMPCs) and hBMSCs from the same donors and compared their osteogenic capacity both in vitro and in vivo. After osteogenic induction in monolayer cultures, hMPCs resulted in more robust mineralization and expressed higher mRNA levels of BMP-2, osteopontin and osteocalcin than hBMSCs. Eight weeks after implantation of cellular-β-TCP scaffolds in immunodeficient mice, hMPC implantation showed higher neovascularization and higher percentage of mature bone formation than hBMSC implantation. In conclusion, hMPCs represent a promising cellular candidate for bone tissue engineering.