The objective of this retrospective study was to determine an optimal time point for vitrification of cleavage-stage human embryos. This study included patients who were undergoing day 2 or day 3 vitrified–warmed cleavage-stage embryo transfer at the In Vitro Fertilization (IVF) Programme of the Shanghai First Maternity and Infant Hospital, China, affiliated to the Tongji University School of Medicine, from April 2010 to March 2012. Intervention was made for the entire cohort of vitrified embryos for poor responder patients so as to avoid severe ovarian hyperstimulation syndrome. Embryo survival rate (SR) after vitrification–warming, implantation rate (IR), and clinical pregnancy rate (CPR) were the main outcome measurements. In total, 380 vitrified–warmed cleavage-stage embryo transfer (VWT) cycles were included. We found that the SR after vitrification and warming for day 2 embryos and day 3 embryos were 92.7% and 92.8%, respectively. For poor ovarian responders, the IR of day 2 and day 3 vitrified–warmed embryos was 6.4% and 13.2%, respectively (P = 0.186). The CPR for day 3 vitrified–warmed embryos was significantly higher than that of day 2 vitrified–warmed embryos (17.6 vs. 4.0 % per transfer cycle, P = 0.036). For patients who had their entire cohort of embryos vitrified to prevent severe ovarian hyperstimulation syndrome (OHSS), the IR and CPR were not significantly different for day 2 and day 3 vitrified–warmed embryo transfer. In conclusion, for vitrified–warmed embryo transfer, cryopreservation of the entire cohort of embryos on day 3 resulted in better clinical outcomes compared with cryopreservation on day 2. Therefore, it is highly recommended that cleavage-stage embryos should be vitrified on day 3, but not on day 2, particularly for poor ovarian responder patients.