Objective To explore whether Helq deletion affect the pluripotency of stem cells. Methods Helq knockout embryonic stem cells were obtained by CRISPR-Cas9 gene editing technique.Results The results of immunofluorescence analysis showed that the expression of Oct4 and Nanog had no obvious difference to that of the control cells. The Helq^-/- embryonic stem cells could produce viable pups by tetraploid complementation, indicating that their pluripotency was not affected. Meanwhile, we found that day 2 epiblast-like cells also were obtained through differentiation of the Helq^-/- embryonic stem cells in vitro. Immunostaining and real-time PCR analysis showed that the gene expression of Helq^-/- epiblast cells were similar to the wild type cells. Conclusions Taken together, it is proved that the genomic instability caused by Helq deletion does not affect the pluripotency of pluripotent stem cells.