Fengycin,　as　a　lipopeptide　produced　by　Bacillus　subtilis,　displays　potent　activity　against　filamentous　fungi,　including　Aspergillus　flavus　and　Soft-rot　fungus,　which　exhibits　a　wide　range　of　potential　applications　in　food　industries,　agriculture,　and　medicine.　To　better　clarify　the　regulatory　mechanism　of　fructose　on　fengycin　biosynthesis,　the　iTRAQ-based　proteomic　analysis　was　utilized　to　investigate　the　differentially　expressed　proteins　of　B.　amyloliquefaciens　fmb-60　cultivated　in　ML　(without　fructose)　and　MLF　(with　fructose)　medium.　The　results　indicated　that　a　total　of　811　proteins,　including　248　proteins　with　differential　expression　levels　(162　which　were　upregulated　(fold　＞　2)　and　86,　which　were　downregulated　(fold　＜　0.5)　were　detected,　and　most　of　the　proteins　are　associated　with　cellular　metabolism,　biosynthesis,　and　biological　regulation　process.　Moreover,　the　target　genes＇　relative　expression　was　conducted　using　quantitative　real-time　PCR　to　validate　the　proteomic　analysis　results.　Based　on　the　results　of　proteome　analysis,　the　supposed　pathways　of　fructose　enhancing　fengycin　biosynthesis　in　B.　amyloliquefaciens　fmb-60　can　be　summarized　as　improvement　of　the　metabolic　process,　including　cellular　amino　acid　and　amide,　fatty　acid　biosynthesis,　peptide　and　protein,　nucleotide　and　nucleobase-containing　compound,　drug/toxin,　cofactor,　and　vitamin;　reinforcement　of　peptide/protein　translation,　modification,　biological　process,　and　response　to　a　stimulus.　In　conclusion,　this　study　represents　a　comprehensive　and　systematic　investigation　of　the　fructose　mechanism　on　improving　fengycin　biosynthesis　in　B.　amyloliquefaciens,　which　will　provide　a　road　map　to　facilitate　the　potential　application　of　fengycin　or　its　homolog　in　defending　against　filamentous　fungi.