To genome-widely mine pathogenesis-related genes of Xanthomonas oryzae pv. oryzicola (Xoc), which is the casual agent of bacterial leaf streak resulting in significant yield loss and poor quality in rice, a Tn5 transposon-mediated mutation library was generated. Twenty-five thousand transformants were produced by using Tn5 transposome, appropriately corresponding to 5 × ORF coverage of the genome, and inoculated into rice and tobacco, individually and respectively, for screening candidate virulence genes. Southern blot and thermal asymmetric interlaced polymerase chain reaction analysis of Tn5 insertion sites of randomly selected mutants suggested a random mode of transposition and a saturation library. Characterization of extracellular polysaccharides, extracellular protease activity, and pigment production of individual mutants in the growth media revealed that 11 mutants enhanced in growth, 12 reduced extracellular polysaccharide production, 12 lost extracellular protease activity completely or partially, and 21 were pigment deficient. In planta pathogenicity assays revealed 253 mutants reduced virulence in rice, but kept triggering hypersensitive response in tobacco; 49 lost the ability to elicit HR in tobacco and pathogenicity in rice; and 3 still induced hypersensitive response in tobacco, but lost pathogenicity in rice. The achieved mutant library of Xoc is of high-quality and nearly saturated and candidate virulence mutants provided a strong basis for functional genomics of Xoc.