Lysine acetylation (Kac), a recently discovered post-translational modification, plays a key role in the regulation of diverse cellular processes.Fusarium oxysporum is a destructive necrotrophic fungal pathogen distributed worldwide with broad ranging hosts. However, the functions of Kac are unknown in FO. or any other plant fungal pathogens. Here, we comprehensively evaluated the acetylation proteome of FO. Our results show that although the acetylation were largely conserved, different organisms contained distinct crotonylated proteins with unique functions. Bioinformatic analysis demonstrated that the majority of crotonylated proteins were distributed in cytoplasm, mitochondria and nucleus). The identified proteins were found to be involved in various metabolic and cellular processes, such as cytoplasmic translation and structural constituent of ribosome. Protein interaction network analysis demonstrated that a mass of protein interactions are regulated by acetylation. These data represent the first report of the crotonylome of FO. and provide a good foundation for further explorations of the role of Kac in plant fungal pathogens.