To investigate the suitable timing to transfer blastocysts vitrified on day 6 in frozen-thawed cycles. Retrospective analysis. This is a retrospective cohort study of 1788 frozen-thawed cycles of blastocysts vitrified either on day 5 or on day 6 and transferred between June 2017 and November 2018. There were 518 cycles included blastocysts vitrified on day 6 (Group A) and 1270 cycles included blastocysts vitrified on day 5 (Group B). According to the timing for blastocyst transfer which was 5 or 6 days after ovulation or progesterone use in hormone replacement therapy (HRT) cycle, the cycles in Group A were divided into two groups: cycles with blastocysts transferred on day 5 (Group C, 103 cycles) and cycles with blastocysts transferred on day 6 (Group D, 415 cycles). Compared with Group A, the female patients in Group B was younger(31.37 ±4.42 VS 31.95 ±4.63, P<0.05). There was no significant difference in male age, thickness of endometrium, endometrial preparation methods and the proportion of primary infertility patients between Group A and Group B. The rate of single blastocyst transfer (SBT), clinical pregnancy rate(cPR) and implantation rate in Group B were significantly higher than those in Group A (84.2% VS 65.8%, 66.0% VS 40.9%, 62.1% VS 35.1%, P<0.001), and the early miscarriage rate and multiple pregnancy rate in Group B were significantly lower than those of Group A (11.2% VS 17.9%, 8.9% VS 15.1%,P<0.01). The cPR and implantation rate in Group C were significantly higher than those in Group D(55.3% VS 37.3%,44.8% VS 32.6%, P<0.01). No significant differences were found between Group C and Group D in terms of early miscarriage rate and multiple pregnancy rate. The rate of SBT, cPR and implantation rate in Group B were significantly higher than those in Group C (84.2% VS 61.2%,66.0% VS 55.3%,62.1% VS 44.8%, P<0.05), and the early miscarriage rate in Group B was significantly lower than that of Group C (11.2% VS 21.1%,P<0.05). Transfer the blastocysts on 5 days, instead of 6 days after ovulation or progesterone use in HRT cycle, could improve the cPR and implantation rate of the blastocysts vitrified on day 6 in frozen-thawed cycles. The cPR and implantation rate of blastocysts vitrified on day 5 are significantly higher compared with blastocysts vitrified on day 6, and the early miscarriage rate is lower, no matter the timing to transfer blastocysts vitrified on day 6.