Mesenchymal stem cells (MSCs) are multipotent cells traditionally derived from bone marrow (BM). They have been demonstrated to be widely applied in tissue regeneration and cellular therapy. As an alternative to BM, an umbilical cord (UC) is considered as a potential source of MSCs. Here, we showed that human UC-MSCs were easily isolated by a single enzymatic digestion and characteristic of plastic adherence and fibroblast-like morphology. UC-MSCs isolation was successful in 15 of 15 samples. The colony-forming unit-fibroblast frequency was obtained 54 ± 1.33 from 10³ UC-MSCs at passage 3, and the doubling time was (24.15 ± 0.49) h. Almost 10¹⁰ UC-MSCs were largely produced in about 30 days. By flow cytometry analysis, the adherent cells displayed an abundant presence of CD73, CD90 and CD105 and absence of CD34, CD45 and HLA-DR. When cultured in differentiation media, they can be differentiated into adipocytes, osteocytes and chondrocytes. RT-PCR reactions confirmed that their multidifferentiation related genes were positive. Moreover, stem cell-related transcription factors Nanog, Oct-4 and Sox-2 were positively expressed in UC-MSCs. On the basis of these findings, the single enzyme method is a good method to obtain large-scale production of MSCs from whole human UC in a short time, and the UC can be considered as a novel and convenient source of adult MSCs displaying high expansion potential and primitive pluripotent stem cells.