<p>Embryo cryopreservation is widely used in infertility treatments; however, many questions remain about the risks of vitrification for newborns. This study was designed to determine the impacts of vitrification and re-vitrification of human blastocysts on the <i>H19/IGF2</i>-DMR methylation status. Top-quality donor embryos were carefully selected for this research. These embryos were cultured until they reached the blastocyst stage and were subsequently divided into three separate groups: 1) fresh group as the control, 2) vitrified, and 3) re-vitrification group. There were three embryos in each group. Then, the methylation levels of <i>H19/IGF2</i>-DMRs of the three blastocyst groups were separately assessed using the bisulfite sequencing PCR (BSP) method. The findings indicated that the total methylation levels in the control group, vitrification group, and re-vitrification group were 42.71%, 40.90%, and 39.23%, respectively. There were no notable differences between the groups (P value &gt; 0.05). This investigation revealed that re-vitrification had no detrimental effects on the methylation status of the <i>H19/IGF2</i>-DMR locus in human blastocysts. Based on the findings, embryo cryopreservation and re-cryopreservation did not have a negative impact on the epigenetic landscape. However, more research is needed to find possible consequences of re-cryopreservation on epigenetic errors in low-quality embryos and embryos originating from infertile couples.</p>