Histones are the main components in chromatin organization, and the protein levels of histones significantly affect chromatin assembly. However, how the protein levels of histones are regulated, especially whether and how histones are degraded, is largely unclear. Here, we found that histone H2B is mainly degraded through the proteasome mediated pathway, and the lysine 120 site of H2B is essential for the K48-linked polyubiquitination and protein degradation of H2B. Moreover, our results further indicated that the degradation-impaired H2BK120R mutant shows an increased nucleolus localization, and inhibition of the proteasome results in an elevated nucleolus distribution of wild-type H2B, which is similar to that of H2BK120R mutants. More importantly, our in vitro data showed that only the nucleolus fractions can ubiquitinate and degrade the purified H2B, suggesting that the nucleolus, in addition to its canonical roles in the regulation of ribosome genesis and protein translation, is likely associated with H2B protein degradation. Therefore, these findings revealed a novel mechanism for the regulation of H2B protein degradation in which a nucleolus associated proteasome pathway is involved.