Since the discovery of post-translational modification (PTM) by the small ubiquitin-related modifiers (SUMOs), a multitude of proteins have been described to be reversibly modified, resulting in the alteration of several cellular pathways. Interestingly, various pathogens gain access to this modification system, although the molecular mechanisms and functional consequences are barely understood. We show here that the adenoviral oncoprotein E1B-55K is a substrate of the SUMO conjugation system, which is directly linked to its C-terminal phosphorylation. This regulative connection is indispensable for modulation of the tumor suppressor p53/chromatin-remodeling factor Daxx by E1B-55K and, consequently, its oncogenic potential in primary mammalian cells. In virus infection, E1B-55K PTMs are necessary for localization to viral transcription/replication sites. Furthermore, we identify the E2 enzyme Ubc9 as an interaction partner of E1B-55K, providing a possible molecular explanation for SUMO-dependent modulation of cellular target proteins. In conclusion, these results for the first time provide evidence how E1B-55K PTMs are regulated and subsequently facilitate exploitation of the host cell SUMOylation machinery.