Protein kinase A (PRKACA) has been documented as a pivotal regulator in meiosis and mitosis arrest. Although our previous work has established that PRKACA regulates cell cycle progression of mouse fertilized eggs by inhibiting M-phase promoting factor (MPF), little is known about the intermediate factor between PRKACA and MPF in the mitotic cell cycle. In this study, we investigated the role of the PRKACA/CDC25B pathway on the early development of mouse fertilized eggs. Overexpression of unphosphorylatable CDC25B mutant (Cdc25b-S321A or Cdc25b-S229A/S321A) rapidly caused G₂-phase eggs to enter mitosis. Microinjection of either Cdc25b-WT or Cdc25b-S229A mRNA also promoted G₂/M transition, but much less efficiently than Cdc25b-S321A and Cdc25b-S229A/S321A. Moreover, mouse fertilized eggs overrode the G₂ arrest by microinjection of either Cdc25b-S321A or Cdc25b-S229A/S321A mRNA, which efficiently resulted in MPF activation by directly dephosphorylating CDC2A-Tyr15, despite culture under conditions that maintained exogenous dibutyryl cAMP. Using a highly specific antibody against phospho-Ser321 of CDC25B in Western blotting, we showed that CDC25B-Ser321 was phosphorylated at the G₁ and S phases, whereas Ser321 was dephosphorylated at the G₂ and M phases in vivo. Our findings identify CDC25B as a potential target of PRKACA and show that PRKACA regulates G₂/M transition by phosphorylating CDC25B-Ser321 but not CDC25B-Ser229 on the first mitotic division of mouse fertilized eggs..