Objective To study if the umbilical cord mesenchymal stem cells of cryopreservation could be induced to differentiated into osteoblasts and immunohistochemical evaluation. Methods Totally 306 cases of healthy fetal umbilical cord were collected and frozen. Mesenchymal stem cells were isolated from the Wharton’s jelly of human umbilical cord tissue by the tissue explant adherent method. Morphology of primitive cells was observed by inverted microscopy. Immunophenotypes and cell cycle of umbilical cord mesenchymal stem cells were measured using immunohistochemical analysis. Umbilical cord mesenchymal stem cells were thawed and subcultured to passage 10. Upon induction with osteogenic inductive medium， the osteogenic ability of passage 10 of umbilical cord mesenchymal stem cell was evaluated by calcium nodules， the immunofluorescent analysis of bone sialoprotein and the assay of alizarin red staining separately. Results Immunohistochemical analysis showed that the cultured cells expressed high levels of the mesenchymal stem cells surface markers CD73, CD90 and CD105, but did not express hematopoietic cells surface markers CD34 and CD45. The survival rate of umbilical cord mesenchymal stem cells after resuscitation was 90%. The cell cycle analysis indicated that 80% of the cells of passage 10 were in G₀/G₁ phase and 20% in S+G₂/M phase. Passage 10 cells treated with osteogenic inductive medium displayed a higher calcium nodule expression compared with control cells. Moreover, the cells, induced in osteogenic inductive medium, were positive for osteocalcin and bone sialoprotein staining and formed the mineralized nodules. Conclusion Umbilical cord mesenchymal stem cells can be induced into osteoblasts with osteogenic inductive medium and. still maintain their highly self-proliferation and multi-directional differentiation.