The pEg3 protein is a member of the evolutionarily conserved KIN1/PAR-1/MARK kinase family which is involved in cell polarity and microtubule dynamics. In Xenopus, pEg3 has been shown to be a cell cycle dependent kinase whose activity increases to a maximum level during mitosis of the first embryonic cell division. CDC25B is one of the three CDC25 phosphatase genes identified in human. It is thought to regulate the G2/M progression by dephosphorylating and activating the CDK/cyclin complexes. In the present study we show that the human pEg3 kinase is able to specifically phosphorylate CDC25B in vitro. One phosphorylation site was identified and corresponded to serine 323. This residue is equivalent to serine 216 in human CDC25C which plays an important role in the regulation of phosphatase during the cell cycle and at the G2 checkpoint. pEg3 is also able to specifically associate with CDC25B in vitro and in vivo. We show that the ectopic expression of active pEg3 in human U2OS cells induces an accumulation of cells in G2. This effect is counteracted by overexpression of CDC25B. Taken together these results suggest that pEg3 is a potential regulator of the G2/M progression and may act antagonistically to the CDC25B phosphatase.