Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial leaf blight, a serious disease of rice worldwide. A Tn5-based transposon randomly insertional mutant library was previously constructed. By screening mutants against susceptible rice cultivar IR24, four mutants were identified with reduced virulence on rice plants and were found to have Tn5 transposon inserted at an endo-1,4-beta-D glucanase (E.C. 3.2.1.4) gene eglXoB. In planta growth analysis indicated that multiplication of the mutants in rice leaves was greatly reduced comparing to the Xoo wild-type strain. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that the expression of eglXoB was induced in planta. Genetic complementation of these mutants with a functional eglXoB gene restored both virulence and in planta growth, suggesting that the eglXoB gene was required for virulence. Ectopic expression of eglXoB in Escherichia coli demonstrated its endoglucanase activity. Otherwise, the growth of the mutants in synthetic medium containing cellulose as the sole sugar source was not affected. Data of this study suggested eglXoB gene is required for pathogenesis of rice bacterial blight disease.