Updated project metadata. Human cytomegalovirus (HCMV) is an important pathogen that extensively modulates host cells, downregulating >900 human proteins over the course of viral replication and degrading ≥133 proteins shortly after infection. The mechanism of degradation of most host proteins remains unresolved, and the functions of many viral proteins are incompletely characterised. We performed a systematic interactome analysis of 169 canonical HCMV proteins, and a subset of non-canonical HCMV proteins, in infected cells. This identified an extensive network of >3,400 virus-host and >150 virus-virus protein interactions, providing insights into novel functions for multiple viral genes. Domain analysis predicted binding of the viral UL25 protein to the SH3 domains of NCK Adaptor Protein 1. Viral interacting proteins were identified for 31/133 degraded host targets. Finally, the uncharacterised, non-canonical ORFL147C protein was found to interact with elements of the mRNA splicing machinery, and a mutational study suggested its importance in viral replication. The interactome data will be important for future studies of herpesvirus infection.