Proper control of cell cycle progression and barrier function are essential processes to the maintenance of epithelial cell homeostasis. The contribution of tight junction proteins to barrier function is well established, whereas their contribution to cell cycle control is only beginning to be understood. Centrosomes are the principal microtubule organizing centers in eukaryotic cells and centrosome duplication and separation are linked to the cell cycle and mitotic entry. Here we demonstrate that occludin localizes with centrosomes in Madin-Darby canine kidney cells. Immunocytochemistry and biochemical fractionation studies reveal occludin localizes with centrosomes during interphase and occludin Ser-490 phosphorylation at centrosomes increases with mitotic entry. Stable expression of aspartic acid phosphomimetic (S490D) results in centrosomal localization of occludin and increases cell numbers. Furthermore, we provide evidence that occludin regulates centrosome separation and mitotic entry as the nonphosphorylatable alanine mutation (S490A) impedes centrosome separation, delays mitotic entry, and reduces proliferation. Collectively, these studies demonstrate a novel location and function for occludin in centrosome separation and mitosis.