Fungal keratitis caused by Aspergillus flavus (A. flavus) can result in severe inflammation and corneal stromal melting, leading to visual impairment. This study aimed to identify virulent genes correlated with the severity of A. flavus keratitis using whole-genome sequencing.Whole-genome sequencing of 21 clinical A. flavus strains from cornea was performed to elucidate the pathogenesis of A. flavus in infectious keratitis, followed by pan-genome analysis and virulence analysis. To further understand the results from the previous analyses, growth phenotypes and virulence effect of mutant strains were validated experimentally, including the spore counting, growth pattern under different conditions, and clinical and pathological evaluation of A. flavus keratitis in mice models.The A. flavus pan-genome was composed of 17,326 gene clusters with a core genome of 5378 (31.0% of the pan-genome) orthogroups in all 21 isolates. Virulence gene analysis revealed 183 genes contributing to A. flavus pathogenesis and mutation of the kexB gene was associated with the severity of keratitis. The kexB mutant (ΔkexB) strains showed significantly reduced conidia formation and lower growth rates in the presence of the cell-wall perturbing agents. Further, the mice models validated that clinical score, and corneal perforation rates significantly decreased in the group infected by ΔkexB strains. Infiltration of immune cells, gene expression of cytokines, and matrix metalloproteinase (MMP) were also decreased in the mutant group.The role of kexB gene in A. flavus keratitis was identified through whole-genome sequencing. Its mutation impairs conidia formation, cell wall integrity, and invasion, leading to milder clinical symptoms.