AbstractThe familial cylindromatosis tumour suppressor CYLD contains three cytoskeleton‐associated protein glycine‐rich (CAP‐Gly) domains and a deubiquitinase domain. The tumour‐suppressing function of CYLD has been attributed to its deubiquitinase domain, which removes lysine‐63‐linked polyubiquitin chains from target proteins, leading to the inhibition of cell survival and proliferation. In this study, we have detected an interaction of CYLD with the mitotic kinase Aurora‐B. The interaction is mediated by the third CAP‐Gly domain of CYLD and results in suppression of Aurora‐B activity. Mechanistic studies reveal that the inhibition of Aurora‐B activity by CYLD is independent of its deubiquitinase activity. Instead, CYLD interacts with protein phosphatase 2A (PP2A) and promotes the ability of PP2A to bind and dephosphorylate Aurora‐B at threonine‐232. Cylindromatosis‐associated truncating mutations of CYLD abolish its interaction with PP2A, its enhancing effect on the PP2A/Aurora‐B interaction, and its inhibitory effect on Aurora‐B activity. These findings uncover Aurora‐B and PP2A as novel binding partners of CYLD and suggest that CYLD negatively regulates Aurora‐B activity through acting on the PP2A axis. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.