Fusaricidins produced by Paenibacillus polymyxa are lipopeptide antibiotics with outstanding antifungal activity. In this study, the whole gene cluster responsible for fusaricidin biosynthesis (fusA) was isolated and identified from the cDNA library of one biocontrol agent P. polymyxa SQR-21 (SQR-21). MALDI-TOF MS analysis confirmed that SQR-21 could produce four kinds of fusaricidins: A, B, C, and D. A central promoter that drove the transcription of fusGFEDCBA was revealed by mapping of the fus promoter region by 5' deletions. The disruption of fusA in SQR-21 led to the abolishment of fusaricidin production and antifungal activity. The direct interaction between a potential regulator, AbrB, and the promoter region of fus gene cluster was confirmed by electrophoretic mobility shift assays. One abrB disruption mutant showed significantly higher antifungal activity compared with the wild type. These results revealed a pathway for the transcriptional regulation of the fus gene cluster in P. polymyxa.