Bacillus amyloliquefaciens C06, a potential agent in biological preservation of post-harvest fruit, was found to secrete extra-cellular gamma-polyglutamic acid (gamma-PGA) in liquid culture. In this work, M306, a transposon mutant of B. amyloliquefaciens C06, defective in forming structured colony and displaying enhanced ability of producing gamma-PGA, was obtained. Inverse PCR and quantitative reverse transcription PCR (qRT-PCR) analysis demonstrated that the defective phenotype in M306 was associated with an ORF showing high similarity to RBAM_034550 from B. amyloliquefaciens FZB42. In this paper, the ORF was designated pbrA, standing for gamma-PGA production and biofilm formation regulatory factor. qRT-PCR analysis also indicated that pbrA down-regulated mRNA expression of epsD and yqxM, the crucial genes involved in biofilm formation, but affected little on expression of ywtB, the gene directing gamma-PGA synthesis. Evaluations in gamma-PGA productivity of wild-type C06 and its mutants C06 Delta epsA and C06 Delta tasA, respectively, deficient in producing exopolysaccharides (EPS) and TasA, revealed that gamma-PGA overproduction in M306 was probably due to the redistributed metabolic flux caused by defective production of EPS.