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<b>: </b>The biosynthesis of penicillin G (PG) is compartmentalized, which forces penicillin and its intermediates to cross the membrane barriers. Although many aspects around the penicillin intermediates traffic system remain unclosed, the transmembrane transporter protein involvement has been only predicted. In the present work, detection of PG and isopenicillin N (IPN) in <i>Monascus ruber</i> M7 was performed and functions of <i>mfst</i> gene as a transporter were investigated by the combination of gene deletion (&#916;<i>mfst</i>) complementation (&#916;<i>mfsT</i>::<i>mfsT</i>) and overexpression (M7::PtrpC-<i>mfsT</i>). While, the feeding of PG pathway precursor side chain and amino acids, i.e., phenylacetic acid, D-valine, and L-cysteine was performed for the interpretation of <i>mfsT</i> gene role as an intermediate transporter. The results showed that, the feeding of phenylacetic acid, D-valine, and L-cysteine possessed a significant effect on morphologies, secondary metabolites (SMs) production of all above-mentioned strains including <i>M. ruber</i> M7. The results of UPLC-MS/MS revealed that, &#916;<i>mfsT</i> interrupt the penicillin G (PG) production in <i>M. ruber</i> M7 by blocking the IPN transportation, while PG and IPN produced by the &#916;<i>mfsT</i>::<i>mfsT</i> have been recovered the similar levels to those of <i>M. ruber</i> M7. Conclusively, these findings suggest that the <i>M. ruber</i> M7 is, not only a PG producer, but also, indicate that the <i>mfsT </i>gene is supposed to play a key role in IPN intermediate compound transportation during the PG production in <i>M. ruber</i> M7.