Abstract: Aspergillus flavus can produce the highly toxic secondary metabolite aflatoxin B₁ (AFB₁), which poses a serious threat to food safety 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. The effect of AFLA_105170 gene deletion on the growth phenotype and sclerotia formation of A. flavus was first 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 AFLA_105170 significantly inhibited the growth, spore development, sclerotia production, and AFB₁ production. It was found that AFB₁ yield in the AFLA_105170 gene deletion strain decreased by 69.81% in PDB medium and by 64.56% in PDA medium respectively. Furthermore, RT-PCR results showed that AFB₁ synthesis-related genes, aflJ, aflW, aflS, and aflM were significantly down-regulated in AFLA_105170 deletion strain. 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 AFB₁ biosynthesis by suppressing the expression of genes involved in the AFB₁ synthesis. The results of the study provide a scientific basis for effective prevention and control of A. flavus and aflatoxin.