Abstract: The objective of this study was to investigate the impact of the transcription factor Afndt6 on the growth, development, aflatoxin biosynthesis, and pathogenicity of Aspergillus flavus. In this study, A. flavus strain with Afndt6 gene deletion was constructed by homologous recombination using the selection gene pyrG and identified via diagnosis PCR analysis. The regulatory roles of Afndt6 on cell growth and development, including spore production and conidiophore formation, were investigated by means of stereomicroscope observation. Aflatoxin B₁ production was determined via thin layer chromatography, and the transcriptional level of genes participated in aflatoxin biosynthetic pathway was profiled with qRT-PCR. The medium containing reagents such as congo red (CR) and hydrogen peroxide (H₂O₂) was used to investigated the response ability of A. flavus ΔAfndt6 strain to external stress. Additionally, the pathogenicity of A. flavus ΔAfndt6 strain on peanut and maize seeds was also evaluated. The results showed that compared with the control strains, the growth rate and conidial production of ΔAfndt6 strain had no significant change, but the conidial color of colony became white. The gene deletion strain was insensitive to cell wall stress agent CR and oxidative stress. The growth inhibition rates of control strain and ΔAfndt6 strain were 31.10% and 25.81%, respectively, when treated with 200 μg/mL CR. The growth inhibition rates of control strain and ΔAfndt6 strain were 47.94% and 17.05%, respectively, when treated with 15 mmol/L H₂O₂.The aflatoxin yield of ΔAfndt6 strain was significantly increased, and the infection ability on peanut and corn seeds was enhanced. This study showed that the transcription factor encoded by Afndt6 gene can affect the growth and development of A. flavus and negatively regulate the biosynthesis of aflatoxin, which can provide reference value and theoretical basis for the prevention and control of A. flavus and aflatoxin contamination in the process of grain storage.