The presence of oxidative stress in sperm cryopreservation induces sperm DNA damage. Our previous study has discovered that γH2AX, the DNA-damaged marker, was activated in the early mouse embryos fertilized with hydrogen peroxide (H2O2)-treated sperm. Furthermore, we found that checkpoint proteins ATM and Chk1 were phosphorylated and activated in the early mouse embryos. On the basis of previous researches, we examined the effects of sperm DNA damage on cell cycle arrest in mouse zygotes fertilized with H2O2-treated sperm. Development of fertilized eggs arrested at the PN disappearance stage. At 19 and 24 hours post-insemination (hpi), the percentage of zygotes at the PN disappearance stage was higher in H2O2-treated group compared to the control group. Immunofluorescence staining revealed Phospho-Cdc25C (Ser216) and Phospho-Cdc25B (Ser323) in or surrounding a single pronucleus, following insemination with H2O2-treated sperm. Our study suggests that fertilization with DNA-damaged sperm results in cell cycle arrest mediated by G2/M checkpoint activation in one of the pronuclei in mouse zygotes fertilized with H2O2-treated sperm; Phospho-Cdc25C and Phospho-Cdc25B correlate with activating G2/M checkpoint in zygotes fertilized with H2O2-treated sperm.