Abstract In the previous study, we used genome shuffling to improve fengycin production of the original strain Bacillus amyloliquefaciens ES-2-4. After two rounds of genome shuffling, a high-yield recombinant FMB72 strain that exhibited 8.30-fold increase in fengycin production was obtained. In this study, comparative proteomic analysis of the parental ES-2-4 and genome-shuffled FMB72 strains was conducted to examine the differentially expressed proteins. In the shuffled strain FMB72, 50 differently expressed spots (p < 0.05) were selected to be excised and analyzed using Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry, and finally 44 protein spots were confidently identified according to NCBI database. According to clusters of orthologous groups (COG) functional category analysis and related references, the differentially expressed proteins could be classified into several functional categories, including proteins involved in metabolism, energy generation and conversion, DNA replication, transcription, translation, ribosomal structure and biogenesis, cell motility and secretion, signal transduction mechanisms, general function prediction. Of the 44 identified proteins, signaling proteins ComA and Spo0A may positively regulate fengycin synthesis at transcriptional level. Taken together, the present study will be informative for exploring the exact roles of ComA and Spo0A in fengycin synthesis and explaining the molecular mechanism of fengycin synthesis.