Vitrification has been suggested to be an effective method for the cryopreservation of human ES cells. However, the efficiency of vitrification with different vehicles remains a matter of ongoing controversy. The objective of this study was to assess the efficiency of cryopreservation in human ES cells by vitrification using different vehicles. A human ES cell line and a variety of vehicles, including micro-droplet (MD), open-pulled straw (OPS) and electron microscopic grid (EM-grid), were employed in an attempt to assess vitrification efficiency. In order to evaluate the survivability and the undifferentiated state of the post-vitrified human ES cells, we conducted alkaline phosphatase staining and characterization via both RT-PCR and immunofluorescence assays. The survival rates of the post-vitrified human ES cells using MD, OPS and EM-grid were determined to be 61.5%, 66.6% and 53.8%, respectively. There also exist significant differences between slow-freezing and vitrification (p<0.01). However, no significant differences were detected between the vehicle types. Finally, the pluripotency of human ES cells after thawing was verified by teratoma formation. Cryopreservation using vitrification is more effective than slow-freezing, and the efficiency of vehicles proved effective with regard to the preservation of human ES cells.