Update information. Macrolactin is a compound with complex macrolide structure which only be obtained through microbiological fermentation now. It has excellent antifungal, antibacterial and antitumor activity. In this study, Bacillus siamensis YB304 was used as the research object, and a mutant Mut-K53 with stable genetic characters was selected by UV-ARTP compound mutation. The macrolactin yield was 156.5 mg/L, 395% higher than original strain. The metabolic pathway changes and the regulatory mechanism of macrolactin were analyzed by quantitative proteomics combined with parallel reaction monitoring. The results showed that the utilization of carbon sources such as sucrose was enhanced in Mut-K53; the glycolysis pathway was positively up-regulated; the amino acid metabolism pathway such as aromatic amino acids was significantly up-regulated. The production of various precursor substances such as acyl-CoA and amino acids was one of the main reasons for the increase of macrolactin production. At the same time, the key proteins of macrolactin biosynthesis are mostly up-regulated, which is the direct reason for the increase of macrolactin production.