A quantitative phosphoproteome was conducted to explore the phosphorylation changes and host cellular responses upon PRV infection. Besides, the characteristics of the phosphorylated proteins were analyzed, including molecular distribution, percentage of the phosphorylated (STY) sites, and phosphorylated motifs. Additionally, the GO and KEGG analyses of the significantly changed phophoproteins were performed. Finally, DNMT1, HDAC6, EGFR, GRK2, VIM, and G3BP1 were chosen for analysis of their effect on PRV replication using an inhibitory-based experiment, and the results showed that all these proteins are necessary for efficient PRV replication.