Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. This study identified and characterized the contribution of the twin-arginine translocation (Tat) pathway to motility, chemotaxis, extracellular polysaccharide (EPS) production and virulence in X. oryzae pv. oryzae strain PXO99. The tatC disruption mutant (strain TCM) of strain PXO99 were generated, and confirmed both by PCR and Southern blotting. Strain PXO99 cells were highly motile in NYGB 0.3% soft agar plate. In contrast, the tatC mutation impaired motility. Furthermore, strain TCM cells lacked detectable flagella and exhibited almost no chemotaxis toward glucose under aerobic conditions, indicating that the Tat secretion pathway contributed to flagellar biogenesis and chemotactic responses. It was also observed that strain TCM exhibited a reductive production of extracellular polysaccharide (EPS) and a significant reduction of virulence on rice plants when compared with the wild type PXO99. However, the tatC mutation in strain PXO99 did not affect growth rate and the ability to induce hypersensitive response (HR) in nonhost tobacco (Nicotiana tabacum L. cv. Samsun). Our findings indicated that the Tat system of X. oryzae pv. oryzae played an important role in the pathogen's virulence.