We have identified the most appropriate method of isolating human umbilical cord matrix-derived mesenchymal stem cells (UCM-MSCs) and compared morphological, phenotypic, proliferative and differentiation characteristics of UCM-MSCs with bone marrow-derived MSCs (BM-MSCs) and umbilical cord blood-derived MSCs (UCB-MSCs). Three explant culture methods and three enzymatic methods were compared with regards to time for primary culture, cell number, cell morphology, immune phenotype and differentiation potential. Morphological, phenotypic, proliferative and differentiation characteristics of UCM-MSCs, BM-MSCs and UCB-MSCs were also compared. UCM-MSCs isolated using the 10 mm size tissue explant method led to shorter primary culture time, higher numbers of isolated cells and higher proliferation rates compared with other isolation methods. Immune phenotype and multilineage differentiation capacity did not differ significantly among six groups. UCM-MSCs had similar characteristics as BM-MSCs and UCB-MSCs, including fibroblastic morphology, typical immunophenotypic markers and multilineage differentiation capacity. In comparison with UCB-MSCs and BM-MSCs, UCM-MSCs have higher proliferative capacity, higher rate of chondrogenic differentiation, and higher expression of CD 146. The results suggest that the 10 mm size tissue culture method is the optimal protocol for the isolation of UCM-MSCs. Given the distinct advantages of UC, such as accessibility, painless acquisition and abundance of cells obtained, we propose that UC be considered an alternative to BM and UCB as a source of MSCs for cell therapy.