The ability to modulate host cell cycle progression is a requirement of many human viruses in order to facilitate their replication and propagation. Nuclear-replicating DNA viruses frequently stall the host cell cycle in G1 to avoid competition with host DNA replication. Among these viruses is human cytomegalovirus (HCMV), a prevalent beta-herpesvirus. Here, we discover a pro-viral mechanism that employs the deacetylase activity of the human enzyme sirtuin 2 (SIRT2) for HCMV-mediated cell cycle dysregulation. First, we show that the SIRT2 deacetylase activity supports an early stage of HCMV replication. Focusing on these early infection time points, we next define temporal SIRT2 protein interactions and deacetylation substrates by using mass spectrometry-based interactome and acetylome analyses. We find that SIRT2 interacts with and modulates the acetylation level of cell cycle proteins during infection, including the cyclin-dependent kinase 2 (CDK2). Using flow cytometry, cell sorting, and functional assays, we demonstrate that SIRT2 regulates CDK2 K6 acetylation and the G1- to S-phase transition in a manner that supports HCMV replication. Altogether, our findings expand the understanding of mechanisms underlying HCMV-induced cell cycle dysregulation and point toward regulatory feedback between SIRT2 and CDK2 that can have implications in other viral infections and human diseases. This study expands the growing understanding that protein acetylation is a highly regulated molecular toggle of protein function in both host anti-viral defense and viral replication. We describe a pro-viral role for the human enzyme SIRT2, showing that its deacetylase activity supports HCMV replication. By integrating quantitative proteomics, flow cytometry cell cycle assays, microscopy, and functional virology assays, we investigate the temporality of SIRT2 functions and substrates. We identify a pro-viral role for the SIRT2 deacetylase activity via regulation of CDK2 K6 acetylation and the G1-S cell cycle transition. These findings highlight a link between viral infection, protein acetylation, and cell cycle progression.