Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate the roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant Xf Delta pilG and complemented strain Xf Delta pilG-C were generated. While all strains had similar growth curves in vitro, Xf Delta pliG showed significant reduction in cell-matrix adherence and biofilm production compared with wild-type X. fastidiosa and Xf Delta pilG-C. The genes pilE, pilU, pilT, and pilS were down-regulated in Xf Delta pliG when compared with its complemented strain and wild-type X. fastidiosa. Finally, no Pierce's disease symptoms were observed in grapevines inoculated with Xf Delta pilG, whereas grapevines inoculated with the wild-type X. fastidiosa and complemented strain of Xf Delta pilG-C developed typical Pierce's Disease (PD) symptoms. The results indicate that pilG has a role in X. fastidiosa virulence in grapevines.