Neuroblastoma (NB) is the most common solid malignancy in childhood and its prognosis is still generally poor. In contrast to many other cancers, mutations of the p53 tumor suppressor are rare. Instead, significant cytosolic sequestration of wtp53 is one of several mechanisms that attenuate p53 function in this cancer. Here, we report that aberrant p53 hyperubiquitylation contributes to p53 cytoplasmic sequestration in NB. NB lines constitutively harbor an elevated portion of wtp53 as stable ubiquitylated species confined to the cytoplasm. p53 hyperubiquitylation is not due to dysregulation by Hdm2 or proteasomal dysfunction. Instead, the defect lies in p53 regulation by HAUSP, a major p53-deubiquitylating enzyme. In contrast to non-NB cancer cells with nuclear p53 and normal ubiquitylation, p53 from NB cells shows impaired HAUSP interaction. Conversely, interference with p53 hyperubiquitylation in NB cells by Nutlin 3a or by a C-terminal p53 peptide (aa 305-393) results in p53 relocalization from the cytoplasm to the nucleus, and in case of Nutlin, in reactivation of p53's transcriptional and apoptotic functions. Moreover, nutlin and camptothecin act synergistically in inducing NB cell apoptosis. Hence, this study strengthens the rationale for targeting p53 deubiquitylation by drugs like Nutlin as a promising new strategy in NB therapy.