p27(Kip1), a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, is now known as a multifunctional protein that plays crucial roles in cell architecture and migration by regulating rearrangements of the actin cytoskeleton and microtubules. The intracellular level of p27(Kip1) is increased by anti-proliferative stimuli, such as mitogen deprivation and contact inhibition, which also induce formation of primary cilia, microtubule-based membranous organelles that protrude from the cell surface. However, it remains unknown whether p27(Kip1) is associated with ciliogenesis. Here, we have generated p27(Kip1)-knockout hTERT-immortalized human retinal pigment epithelial cells, and found that ciliogenesis is almost completely disrupted in p27(Kip1)-knockout cells. The defect of ciliogenesis is rescued by the exogenous expression of wild-type p27(Kip1) and, surprisingly, its 86-140 amino acid region, which is neither responsible for CDK inhibition nor remodeling of the actin cytoskeleton and microtubules. Moreover, transmission electron microscopy and immunofluorescence analyses reveal that p27(Kip1) abrogation impairs one of the earliest events of ciliogenesis, docking of the Ehdl-associated preciliary vesicles to the distal appendages of the basal body. Our findings identify a novel CDK-independent function of p27(Kip1) in primary cilia formation. (C) 2020 Elsevier Inc. All rights reserved.