Mesenchymal stem cells (MSCs) from adult exhibit self-renewal and multilineage differentiation capacities, making the MSCs promising candidates for cell therapy and tissue engineering. Although bone marrow (BM) is the most universal source of MSCs, other tissues may also contain MSCs. Peripheral blood (PB), in particular, arises as the most attractive source of MSCs due to easy accessibility and noninvasive procedure. However, it is not certain that PB-MSCs have the equal biological characteristics to those of BM-MSCs. The purpose of this study was to compare the biological characteristics between BM-MSCs and PB-MSCs. We adopted granulocyte colony-stimulating factor combined with CXCR4 antagonist AMD3100 to stimulate MSCs to release into blood circulation of the rats. PB-MSCs were obtained from mobilized PB and expanded in long-term culture. BM-MSCs were isolated from the femur and tibia medullary canal of the same rats by density gradient centrifugation. After cell expansion in vitro, cell surface markers and multipotentiality analysis were performed to identify MSCs. Apoptosis resistance to H(2)O(2)-induced apoptosis, proliferation kinetics, cellular senescence, and karyotype analysis were measured to compare the biological characteristics of PB-MSCs and BM-MSCs. PB-MSCs with the typical adherent fibroblast-like morphology were similar to that of BM-MSCs. Both PB-MSCs and BM-MSCs were positive for CD44 and CD90, and negative for CD34 and CD45. They both exhibited trilineage differentiation potential and expressed lineage-specific genes. Although the BM-MSCs showed stronger osteogenic and adipogenic differentiation, PB-MSCs displayed a more chondrogenic capacity. Further, BM-MSCs have greater proliferation ability. Apoptosis resistance and cellular senescence were similar in MSCs derived from both sources. The results of our study demonstrate that PB-MSCs have similar biological characteristics to those of BM-MSCs despite certain minor differences, suggesting PB as a possible alternative source for MSCs.