During exit from mitosis in Xenopus laevis egg extracts, the AAA+ ATPase Cdc48/p97 (also known as VCP in vertebrates) and its adapter Ufd1–Npl4 remove the kinase Aurora B from chromatin to allow nucleus formation. Here, we show that in HeLa cells Ufd1–Npl4 already antagonizes Aurora B on chromosomes during earlier mitotic stages and that this is crucial for proper chromosome segregation. Depletion of Ufd1–Npl4 by small interfering RNA (siRNA) caused chromosome alignment and anaphase defects resulting in missegregated chromosomes and multi-lobed nuclei. Ufd1–Npl4 depletion also led to increased levels of Aurora B on prometaphase and metaphase chromosomes. This increase was associated with higher Aurora B activity, as evidenced by the partial resistance of CENP-A phosphorylation to the Aurora B inhibitor hesperadin. Furthermore, low concentrations of hesperadin partially rescued chromosome alignment in Ufd1-depleted cells, whereas, conversely, Ufd1-depletion partially restored congression in the presence of hesperadin. These data establish Cdc48/p97–Ufd1–Npl4 as a crucial negative regulator of Aurora B early in mitosis of human somatic cells and suggest that the activity of Aurora B on chromosomes needs to be restrained to ensure faithful chromosome segregation.