Aspergillus flavus contaminates crops during preharvest and post-harvest periods and produce carcinogenic mycotoxin aflatoxins posing severe threat to food safety and human health. Lysine 2-hydroxyisobutyrylation is one of the most important reversible post-translational modifications and plays a vital regulatory role in various cellular processes. In order to explore the potential roles of lysine 2-hydroxyisobutyrylation in aflatoxin biosynthesis, protein 2-hydroxyisobutyrylation analysis of A. flavus was performed, and a total of 7156 2-hydroxyisobutyrylation sites in 1473 proteins were identified.