The Burkholderia pyrrocinia Lyc2 strain isolated from healthy plant rhizosphere showed significant antimicrobial activities against a variety of plant pathogens. In this study, a random mutation library was constructed using an EZ-Tn5 transposome kit and Erwinia amylovora was used as an indicator to screen for mutants with defective antibacterial activity. The transposon gene was verified in the chromosome of the Lyc2 strain using polymerase chain reaction (PCR). The gene that was disrupted by transposon was amplified by rescue cloning for functional and bioinformatics analyses. Antibacterial analysis indicated that the mutant Lyc2-MT2918 was defective in antibacterial activity. Sequence alignment of the mutant suggested that the disrupted gene Glu-2918 was homologous to the glutathione (GSH) synthase gene Bamb-2918 of strain B. ambifaria AMMD. Genetic functional analysis and complementary assay of the disrupted gene, which was predicted to encode GSH synthase, indicated the essential role of the Glu-2918 gene in the antibacterial activity of strain Lyc2.