Lysine crotonylation is a common occurrence in eukaryotic cells, regulating various physiological functions, including chromatin remodeling, cellular growth, and development. However, its involvement in viral infections has rarely been documented. In this study, we reveal that pseudorabies virus (PRV) infection significantly alters the global lysine crotonylation levels in porcine kidney PK-15 cells. Specifically, we identified a few viral proteins, including UL54, gM, gD, UL19, UL37, and UL46, which undergo crotonylation modification. Our observations indicate that at 20 h post-infection (hpi), 551 crotonylation sites were reduced across 345 proteins, while 47 new sites emerged in 37 proteins compared to the control group. By 40 hpi, 263 sites had decreased in 190 proteins, while 389 new sites appeared in 240 proteins. Deeper analysis revealed that the proteins with altered crotonylation levels were primarily involved in binding, catalytic activity, biosynthetic processes, ribosome activity, and metabolic processes. Additionally, our findings underscored the significance of ribosomes and the endoplasmic reticulum (ER), which were enriched with proteins exhibiting altered crotonylation. Overall, our study for the first time offers new insights into the relationship between crotonylation and herpes virus infection, paving the way for future investigations into the role of crotonylation in viral infections.