Cleavage of human cytomegalovirus (HCMV) genomes and their packaging into capsids requires at least seven essential viral proteins, yet it is not completely understood how these proteins cooperate to accomplish this task. Besides the portal protein pUL104 and the terminase subunits pUL51, pUL56, and pUL89, the UL52 protein is also necessary for HCMV genome encapsidation; however, knowledge about pUL52 is scant. In the absence of pUL52, viral concatemers are not cleaved into unit-length genomes and no DNA-filled capsids are observed, yet no viral or cellular proteins interacting with pUL52 have been identified that would explain how pUL52 exerts its essential role in the HCMV infection cycle. In this study, we aimed at a comprehensive definition of pUL52-interacting proteins in infected cells. Using suitable HCMV mutants, we employed three complementary state-of-the-art proteomic approaches, namely biotin ligase-dependent proximity labeling, affinity purification, and cross-linking mass spectrometry. These experiments, combined with thorough validation by immunoblotting, pointed to several viral DNA-associated proteins and key players pivotal for genome encapsidation as interactors of pUL52. The most noticeable direct pUL52 interaction partners were the terminase subunits pUL56 and pUL89 as well as the portal protein pUL104. Hence, we suggest a model of pUL52 function in which pUL52 mediates association of HCMV genomes with the terminase subunits and the capsid portal. Taken together, our data contribute to the understanding of an essential viral process previously recognized as a prominent antiviral target. Disturbing the identified pUL52 interactions may provide a starting point to develop novel antiviral medication. IMPORTANCE: Human cytomegalovirus (HCMV) can evoke severe disease in immunocompromised patients and, moreover, is the most frequent viral cause of malformations in newborns. The virus-specific process of genome cleavage and packaging into capsids has emerged as an Achilles heel in the HCMV life cycle, which can be targeted by novel antiviral drugs, yet the mechanism of viral DNA encapsidation is only partially understood. Here, we report that the essential viral cleavage-packaging protein pUL52 interacts with several HCMV proteins known to be crucial for genome packaging, with the most prominent ones being the terminase complex and the portal protein. These data provide insight into the role of pUL52 during HCMV infection and may lay the basis for the development of additional antiviral substances tackling viral DNA packaging.