Previously, seventeen extracellular polysaccharide-associated mutants of Xanthomonas oryzae pv. oryzicola (Xoc) were acquired from our randomly Tn5-inserted mutant library. To know the Tn5-inserted genes of these mutants and their contribution to EPS production and virulence in rice,in this study, we first identified and characterized the Tn5-targeted genes in these mutants and then inoculated them in susceptible rice for virulence assessment.Tn5 transposon was inserted in genes of the gum, xan and wxoc clusters in the majority of EPS-reduced mutants. Of the EPS-reduced mutants, three were due to the Tn5 insertion in Xoryp_4217, Xoryp 2488 and Xoryp_0918 genes, respectively. In six EPS-increased mutants, three were in mutagenesis in fimO, pilY and xopQ genes, respectively, resulting in higher EPS production than the wild-type strain RS105. Other three were because of the mutation in Xoryp2392, Xoryp_4221 and Xoryp_3511 genes. Interestingly, XocORF-3511 only exists in X. oryzae but not in other Xanthomomas species. Virulence assays in rice showed that the less EPS production by the mutant, the weaker the virulence in rice. However, those mutants in higher EPS production did not increase virulence significantly in rice compared to that by the wild-type strain.Our findings will help further understand the metabolic pathways for EPS synthesis in Xoc and the specific roles of EPS-associated genes in Xoc-rice interactions.