Cdc25 phosphatases are essential regulators of the cell cycle. In mammalian cells, the Cdc25B isoform activates cyclin A- and cyclin B1-containing complexes and is necessary for entry into mitosis. In this report, we characterise the subcellular localisation of Cdc25B by immunofluorescence in combination with RNA interference to identify specific antibody staining. We find that endogenous Cdc25B is mainly nuclear, but a fraction resides in the cytoplasm during the G2 phase of the cell cycle. Cdc25B starts to appear in S-phase cells and accumulates until prophase, after which the protein disappears. We characterise a nuclear export sequence in the N-terminus of Cdc25B (amino acids 54-67) that, when mutated, greatly reduces the ability of Cdc25B to shuttle in a fluorescence loss in photobleaching assay. Mutation of the nuclear export sequence makes Cdc25B less efficient in inducing mitosis, suggesting that an important mitotic function of Cdc25B occurs in the cytoplasm. Furthermore, we find that when cells are exposed to cycloheximide or ultraviolet irradiation, Cdc25B partially translocates to the cytoplasm. The dependence of this translocation event on a functional nuclear export sequence, an intact serine 323 residue (a 14-3-3 binding site) and p38 mitogen-activated protein kinase activity indicates that the p38 pathway regulates Cdc25B localisation in different situations of cellular stress.