The new gene aurT (GenBank accession number:MH247109.1) in the Aureofuscin biosynthetic gene cluster was obtained by molecular cloning technology. And the function and regulatory mechanism of aurT gene on the Aureofuscin biosynthesis were analyzed by high-efficiency expression, gene knockout, high-performance liquid chromatography (HPLC) and bioinformatics technology in order to enhance the fermentation yield of Aureofuscin. The results showed that AurT belonged to the RhtB family regulator and was an amino acid transporter for Aureofuscin biosynthesis. Compared to original strain, the Aureofuscin production in the aurT gene highly expressing strain (♂aurT) was 1.3 times increased, which was reduced by 65% in the aurT gene knockout strain (ΔaurT), but the Aureofuscin production was not completely inhibited. It indicated that the regulation process of aurT on biosynthesis was different from the pathway-specific regulation gene aurJ3M. Four different structural analogues of PI factors (1,2- propylene glycol, glycerin, ethylene glycol, triethanolamine) were compared, and addition 0.4mL glycerin had the best effect on regulating the Aureofuscin biosynthesis. The yield of Aureofuscin produced by ΔaurT strain was 784μg/mL with exogenous addition of glycerin for 96h, which was only 244μg/mL without addition of glycerin. Further study using RT-PCR experiments verified that the addition of glycerol to the wild-type strain increased the transcription level and expression of aurT gene. It indicated that structural analogues of PI factors might bind to specific receptors in the gene cluster and initiate the expression of related genes. Aureofuscin biosynthesis was regulated by aurT gene via mediating the extracellular transport of PI factors.