Objective To investigate the pathogenic role of melioidosin in pathogenicity of Burkholderia pseudomallei (B. pseudomallei). Methods A synthetase gene associated with melioidosin synthesis in B. pseudomallei was inactivated by homologous recombination. Melioidosin producting ability and the virulent effects of the wild-type and mutant B. pseudomallei upon microorganisms (Penicillium spp., Aspergillus niger spp., Streptococcus faecalis spp. and Staphyloccocus aureus spp.), C. elegans and BALB/c mice were compared. Results The mutant B. pseudomallei underwent notable morphology change, lost the ability to form bacterial membrane and the virulent effects to microorganisms, C. elegans and BALB/c mice. Conclusion It suggests that melioidosin is one of the main virulent factors of B. pseudomallei.