The surfactant-like protein Ki-67 mediates the clustering of chromosomes during mitotic exit, which displaces large cytoplasmic molecules from the future nuclear space and thus enables the separation of cytoplasmic and nuclear components before the nuclear envelope reforms. Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation(1). This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores(2). During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix(3). When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores(2). These pores have a size limit of 39 nanometres(4-7), which raises the question of how larger cytoplasmic molecules are cleared from the nucleus. Here we show in HeLa cells that large cytoplasmic components are displaced before nuclear envelope assembly by the movement of chromosomes to a dense cluster. This clustering occurs when chromosomes approach the poles of anaphase spindles, and is mediated by a microtubule-independent mechanism that involves the surfactant-like protein Ki-67. Ki-67 forms repulsive molecular brushes during the early stages of mitosis(8), but during mitotic exit the brushes collapse and Ki-67 promotes chromosome clustering. We show that the exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, our study reveals that chromosome mechanics help to re-establish the compartmentalization of eukaryotic cells after open mitosis.