The Bacterial Fruit Blotch (BFB) caused by Acidovorax avenae subsp. citrulli is a serious disease on watermelon and melon. A strain MH21 was isolated from a diseased melon fruit, and was identified as subsp. A. avenae subsp. citrulli. In this study, the strain MH21 was mutated using transposon Mini-Tn5 to select the mutants defective in their pathogencity on melon seedling. Southern blot analysis revealed that transposon Tn5 generated the random and single-copy insertions in the chromosome of strain MH21. A mutant M543 lost its pathogenicity on melon seedling was selected and the gene disrupted by Tn5 was identified to be the homolog of hrcR gene, which coded a conserved inner-membrane protein of Type Ⅲ secretion system (TTSS) in some well-documented bacterial pathogens. The pot experiments revealed that pathogenicity of M543 could be complemented by plasmid-born wide-type hrcR gene. Additionally, mutant M543 defective in hrcR gene also lost its ability to trigger hypersensitive reaction (HR) on tobacco leaf. The genetics evidence in this report demonstrated that the TTSS is critical for the pathogenicity of subsp. A. avenae subsp. citrulli on melon.