Abstract: Aspergillus niger can produce the potent carcinogenic ochratoxin A (OTA), which brings a serious threat to food security and human health. Therefore, an in-depth study of the regulatory mechanism of OTA biosynthesis contribute to provide scientific basis for investigating new targets for fungal prevention and control. In the study, the AnHog1 gene deletion strain was constructed, and the effects of AnHog1 gene deletion on the growth and development of A.niger were determined first. Then, the effect of AnHog1 gene deletion on OTA biosynthesis was examined using HPLC-FLD. Finally, the effects of AnHog1 gene deletion on the expressions of genes in OTA gene cluster were investigated by fluorescence quantitative PCR (RT-PCR), the tolerance to hydrogen peroxide (H₂O₂) were explored and the ROS levels were further explored by fluorescence staining. The results indicated that knockout of AnHog1 gene did not affect the growth and spore production of A.niger, but significantly inhibited OTA biosynthesis. The OTA yields decreased by 40.8% in the AnHog1 gene deletion strain compared with the control strain. The expression levels of genes pks, p450, nrps, hal and bzip in the OTA synthesis gene cluster of the AnHog1 deletion strain were down regulated by 64.19%, 25.82%, 18.88%, 51.31% and 48.92% respectively compared with the control strain. In addition, the sensitivity of AnHog1 deletion strain to hydrogen peroxide was increased with increasing H₂O₂ concentration, and the AnHog1 deletion strain had elevated levels of ROS in mycelia. These results suggested that deletion of the AnHog1 gene leads to inhibition in OTA synthesis by suppressing the expression of OTA synthesis genes and breaking the balance of oxidative stress.