Abstract: Aflatoxin B₁（AFB₁） is a highly toxic secondary metabolite produced by Aspergillus flavus, which endangers food security and human health. To investigate the effect of the transcription factor AFLA＿105170 on A. flavus, this study successfully constructed a gene deletion strain of AFLA＿105170 using a homologous recombination strategy. First, the effect of AFLA＿105170 gene deletion on the growth phenotype and sclerotia formation of A. flavus was determined. The effect of the AFLA＿105170 gene deletion on AFB₁ biosynthesis was examined using thin-layer chromatography（TLC） and high-performance liquid chromatography（HPLC）. The effect of AFLA＿105170 gene deletion on the expression levels of genes involved in AFB₁ synthesis was further detected by fluorescence quantitative PCR（RT-PCR）. The cell wall and membrane integrity of the AFLA＿105170 mutant were examined by stress response and staining assay. Finally, the effect of AFLA＿105170 gene deletion on the pathogenicity of strains was investigated using peanut and maize as media. The results showed that deletion of the AFLA＿105170 gene significantly inhibited the growth, spore production, sclerotia production of Aspergillus flavus, and severely inhibited the biosynthesis of AFB₁. It was found that the content of AFB₁ in the AFLA＿105170 gene deletion strain decreased by 69.81% and 64.56% in PDB and PDA medium, respectively, and the genes aflJ, aflW, aflS, and aflM related to AFB₁ synthesis were significantly down-regulated. Moreover, the integrity of cell membrane and cell wall in the AFLA＿105170 gene deletion strain were disrupted, and its pathogenicity in peanut and maize was reduced. These results suggest that the deletion of AFLA＿105170 inhibits A. flavus growth by damaging the integrity of the cell wall and membrane, and affects the synthesis of AFB₁ by suppressing the expression levels of AFB₁ synthesis genes. This study provides a scientific basis for effective prevention and control of A. flavus and aflatoxin.