To assess the effect of assisted hatching (AH) site on the clinical outcomes in vitrified-warmed blastocyst transfer cycles. A total of 160 women who underwent vitrified-warmed blastocyst transfer cycles were randomized to either the ICM group (AH performing at the site near the inner cell mess, ICM), or the TE group (AH performing at the site opposite to the ICM). AH with laser zona drilling was performed 20 or 30 min after thawing once the ICM can be detected. Clinical pregnancy rate, implantation rate, live birth rate and the occurrence rate of monozygotic twins (MZT) pregnancy after transfer of these two groups were compared. No significant difference was found in the clinical pregnancy rate (63.8 % vs. 67.5 %), implantation rate (51.7 % vs. 53.6 %) and live birth rate (57.5 % vs. 62.5 %) between the ICM group and the TE group. The occurrence rate of MZT was comparable between the two groups (3.9 % vs. 5.6 %). The site of assisted hatching has no influence on the implantation, pregnancy and live birth rate in human vitrified-warmed blastocyst transfer cycles.