Emerging evidence points to protein acetylation as a nexus at the interface of virus-host interactions. However, global protein acetylation is understudied during infection, and the temporal control and function of acetylation in the context of infection remains poorly understood. Here, we present the first temporal-spatial characterization of acetylation during infection with human cytomegalovirus (HCMV). By using acetyl-peptide immunoaffinity purification, we identified dynamic changes in acetylation events on both cellular and viral proteins over the course of HCMV infection. Our subsequent analyses demonstrated that these acetylations functionally contribute to the progression of infection.