Abstract: Investigating the regulatory mechanism of bHLH-type transcription factor AfldevR on cell development and aflatoxin B₁ (AFB₁) biosynthesis in Aspergillus flavus can provide novel potential targets for preventing and controlling A. flavus growth and AFB₁ contamination during grain storage. Using AfldevR gene deletion (ΔAfldevR) and complementation strains (ΔAfldevR-com) as research subjects, the effects of AfldevR deletion on colony growth and sporulation of A. flavus were characterized by plate culture, section and stereomicroscopy observation. Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were employed to measure AFB₁ content. The influence of AfldevR gene deletion on the expression levels of genes involved in AFB₁ biosynthesis pathway was also explored. PI and DAPI fluorescent dyes were used to detect the integrity of AfldevR cell membrane and nuclei, respectively. The pathogenicity of ΔAfldevR strain was determined using peanut kernel and corn meal as culture substrates. The results indicated that compared with the control strains, AfldevR gene deletion significantly inhibited the growth rate and spore production of A. flavus, damaged the cell nuclei, and increased cell membrane permeability. The amount of AFB₁ biosynthesis decreased significantly, and the transcriptional levels of aflR, aflA, aflC and aflE were significantly down-regulated. Additionally AfldevR gene deletion strain was more sensitive to oxidative stress, and the growth inhibition rates of the control strain and ΔAfldevR strain treated with 20 mmol/L H₂O₂ were 31.09% and 100%, respectively. Compared with wild-type and complementation strains, AFB₁ yield of ΔAfldevR strain was significantly reduced on peanuts, but showed no significant difference on corn meal. In summary, the AfldevR gene positively regulates the growth of A. flavus, the transcription levels of genes involved in AFB₁ biosynthesis, AFB₁ production and tolerance to oxidative stress. This study provides a theoretical support for the development of fungicides targeting AfldevR in A. flavus.