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Aflatoxin, a carcinogenic secondary metabolite produced by <i>Aspergillus flavus</i>, is a significant threat to human health and agricultural production. Histone 2-hydroxyisobutyrylation is a novel post-translational modification that regulates various biological processes, including secondary metabolism. In this study, we identified the novel histone 2-hydroxyisobutyryltransferase Afngg1 in <i>A. flavus</i>, and explored its role in cell growth, development and aflatoxin biosynthesis. <i>Afngg1</i> gene deletion markedly decreased lysine 2-hydroxyisobutyrylation modification of histones H4K5 and H4K8 compared with the control strain. Additionally, <i>Afngg1</i> deletion inhibited mycelial growth of <i>A. flavus</i>, and the number of conidia and hydrophobicity were significantly decreased. Notably, aflatoxin B₁ biosynthesis and sclerotia production were completely inhibited in the Δ<i>Afngg1</i> strain. Furthermore, the pathogenicity of the Δ<i>Afngg1</i> strain infecting peanut and corn grains was also diminished, including reduced spore production and aflatoxin biosynthesis compared with <i>A. flavus</i> control and <i>Afngg1</i> complementation strains. Transcriptome analysis showed that, compared with control strains, differentially expressed genes in Δ<i>Afngg1</i> were mainly involved in chromatin remodelling, cell development, secondary metabolism and oxidative stress. These results suggest that Afngg1 is involved in histone 2-hydroxyisobutyrylation and chromatin modification, and thus affects cell development and aflatoxin biosynthesis in <i>A. flavus</i>. Our results lay a foundation for in-depth research on the 2-hydroxyisobutyrylation modification in <i>A. flavus</i>, and may provide a novel target for aflatoxin contamination prevention.