Abstract: Aspergillus niger is widely used in food industry.However, recent researches demonstrate that several A. niger strains could produce strong carcinogenic toxins such as ochratoxin A(OTA), posing a serious threat to food safety and human health.In order to elucidate the underlying mechanism of OTA biosynthesis in A. niger, the pyrG gene knockout strain and complementation strain in A. niger were constructed in this paper.The effect of pyrG on OTA biosynthesis was examined by HPLC-FLD, different concentrations of uridine were then added, Real-Time quantitative PCR(RT-qPCR)was performed, and the OTA yield of the pyrG knockout strain in corn kernels were evaluated.The results showed that the OTA yield in pyrG knockout strain was decreased by 77.8% and 75.7%, respectively, compared to the wild-type strain and complementation strain.The addition of different concentrations of uridine had no significant effect on OTA production in pyrG knockout strain, indicating that the regulation of OTA production by pyrG was independent of uridine dose.In addition, compared with the wild-type strain, the expression levels of pks, p450, nrps, hal and bZIP genes in pyrG knockout strain decreased by 52.0%, 36.0%, 84.0%, 46.0% and 74.0%.Compared with the complementation strain, the expression levels of those genes decreased by 55.7%, 42.1%, 85.1%, 50.3%, and 76.0%.Besides, we found that deletion of pyrG resulted in almost no OTA yield in A. niger strains cultivated on corn kernels.Therefore, these results implied that deletion of pyrG repressed the expression levels of pks, p450, nrps, hal, and bZIP, and ultimately reduced the OTA biosynthesis in A. niger CBS 513.88.