Using a powerful new model system for interrogating NEK5 signalling and function in breast cancer, we have determined that NEK5 influences not only breast epithelial cell proliferation but also 3D morphogenesis, and provided novel insights via proteomic interrogation that consolidate the role of NEK5 as a mitotic regulator, lend further support to emerging roles for NEK5 in mitochondrial function and DNA repair, and identify novel functions for this kinase in regulating cytoskeletal organization by Rho GTPases and cell–matrix interaction via hemidesmosomes. Here, NEK5 is linked to breast cancer.