Human blastocysts have been successfully cryopreserved and many live births occur each year using blastocysts that have been cryopreserved once. However, there is a paucity of information on re-cryopreservation of human blastocysts developed from frozen embryos. Therefore, we document the data for 27 blastocysts from 15 cycles (12 patients) of warming to review the efficacy of re-cryopreservation by vitrification of human blastocysts developed from frozen-cleaved embryos. A total of 27 surplus human blastocysts developed from frozen-cleaved embryos, obtained from 12 healthy infertile women undergoing IVF treatment between February 2004 and December 2006, were re-cryopreserved by vitrification. Of these, 26 (96%) re-expanded after warming and were transferred into 12 patients (15 cycles). Following transfer, the implantation rate was 35% (9/26) and the pregnancy rate was 47% (7/15). Two pregnancies ended in miscarriage, 5 healthy babies were born in 4 deliveries, and 1 pregnancy is ongoing. Our results suggest that re-cryopreservation by vitrification of human blastocysts developed from frozen-cleaved embryos can be performed without impairing their implantation ability after warming.