Aims: Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that is synthesized by diguanylate cyclase (DGC) with the GGDEF-domain, regulates diverse virulence phenotypes in pathogenic bacteria. Although 11 genes encoding GGDEF-domain proteins have been shown in the genome of Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99 A, the causal pathogen of bacterial blight of rice, however, little is known about their roles in the c-diGMP regulation of virulence in the pathogen. GdpX1, one of the GGDEF-domain proteins in Xoo was investigated in this study to reveal its regulatory function of bacterial virulence expression through genetic analysis. Methods and Results: GdpX1 was functionally characterized in virulence expression through deletion and overexpression analysis. Bioinformatics analysis revealed the GGDEF-domain in GdpX1 was well conserved, indicating it is a putative DGC. Deletion of gdpX1 resulted in significant increases in virulence, exopolysaccharide (EPS) production and flagellar motility. In contrast, overexpression of gdpX1 dramatically reduced these virulence phenotypes. qRT-PCR analysis showed genes related to the type III secretion system (T3SS), EPS synthesis, and flagellar motility, were up-regulated in Delta gdpX1 and down-regulated in the gdpX1-overexpressed strains. In addition, overexpression of gdpX1 promoted biofilm formation and xylanase activity. Conclusion: GdpX1 is the first GGDEF-domain protein functionally characterized in Xoo, which functions as a negative regulator of bacterial virulence via suppression of virulence-related gene transcription. Significance and Impact of the Study: Identification and functional characterization of GdpX1 provided additional insights into molecular mechanisms of c-di-GMP regulation of bacterial virulence expression.