SET8 (also known as PR-Set7/9, SETD8, KMT5A), a member of the SET domain containing methyltransferase family, which specifically catalyzes mono-methylation of K20 on histone H4 (H4K20me1), has been implicated in multiple biological processes, such as gene transcriptional regulation, cell cycle control, genomic integrity maintenance and development. In this study, we used GST-SET8 fusion protein as bait to search for SET8 interaction partners to elucidate physiological functions of SET8. In combination with mass spectrometry, we identified 40 proteins that potentially interact with SET8. DDX21, a nucleolar protein, was further confirmed to associate with SET8. Furthermore, we discovered a novel function of SET8 in the regulation of rRNA transcription.