Fusariumhead blight is a devastating disease that causes significant economic losses worldwide.Fusarium graminearumis a crucial pathogen that requires close attention when controlling wheat diseases. Here, we aimed to identify genes and proteins that could confer resistance toF. graminearum. By extensively screening recombinants, we identified an antifungal gene,Mt1 (240 bp), fromBacillus subtilis330-2. We recombinantly expressedMt1 inF. graminearumand observed a substantial reduction in the production of aerial mycelium, mycelial growth rate, biomass, and pathogenicity. However, recombinant mycelium and spore morphology remained unchanged. Transcriptome analysis of the recombinants revealed significant down-regulation of genes related to amino acid metabolism and degradation pathways. This finding indicated thatMt1 inhibited amino acid metabolism, leading to limited mycelial growth and, thus, reduced pathogenicity. Based on the results of recombinant phenotypes and transcriptome analysis, we hypothesize that the effect ofMt1 onF. graminearumcould be related to the metabolism of branched-chain amino acids (BCAAs), the most affected metabolic pathway with significant down-regulation of several genes. Our findings provide new insights into antifungal gene research and offer promising targets for developing novel strategies to controlFusariumhead blight in wheat.