Abstract: Objective：To investigate the effect of in vitro extended passaging on the phenotype, proliferation and differentiation characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs).Methods: Methods of enzymatic digestion, tissue explantation and combination of the two were compared for isolation of hUCMSCs. Cell morphology, growth curve, CFU-F forming ability, cell cycle, phenotype and differentiation activity of hUCMSCs after expanding over eighteen passages in vitro were comprehensively assessed. Results：Combination of enzymatic digestion and tissue explantation was the best way to successfully isolate abundant hUCMSCs. After long time expansion in vitro, morphology of hUCMSCs changed from elongated and spindled-shape with narrow cytoplasm to flattened-shape with more lamellopoid. Population doubling time (PDT) increased to more than 60 hours and CFU-F forming ability significantly decreased at P18 suggesting the replicative senescence of hUCMSCs. Mitotic frequency showed more cells in G0/G1-phase, indicating the existence of lower percentage of proliferating cells fraction after long time subsequent passages. MSCs markers were consistently expressed during passaging, while pluripotent stem cells markers Oct-4 and SSEA-4 were negatively expressed at late passages. Specific staining and gene expression detection showed attenuated differentiation viability of hUCMSCs at P18.Conclusions：HUCMSCs were MSCs subpopulation with high proliferation and differentiation viability. Although cellular senescence of hUCMSCs was noted after large amount of expansion in vitro, they still possess stable expression of MSCs markers and the ability of multi-differentiation.