Xylella fastidiosa causes Pierce’s disease of grape and other important plant diseases apparently by occluding xylem vessels during endophytic growth in plants. Large populations of this pathogen develop as biofilms within xylem vessels after its introduction via insect vectors in which it also forms an extensive biofilm in the precibarium. Thus X. fastidiosa cells occur in high spatial densities in both colonized plants and in insects and cell-density-dependent traits might strongly influence its behavior. The genomic sequence of X. fastidiosa reveal the presence of genes related to those in the rpf operon of Xanthomonas campestris that regulate expression of virulence traits in a cell density-dependent fashion via the production and recognition of a diffusible fatty acid signal molecule. We show that rpfF is necessary and sufficient for X. fastidiosa to produce a similar signal molecule in X. fastidiosa and that RpfF- mutants can still colonize grape xylem vessels and are hyperviulent to grape, producing more rapid and intense disease symptoms. In contrast, mutants in this locus can no longer colonize the precibarium of sharpshooters and are thus non-transmissible. The identification of a regulatory locus of the pathogen that so strongly controls colonization of insects provides a tool to better understand the process of insect vectoring of bacterial plant pathogens and suggests new methods of disease control based on altering pathogen behavior in plants and insects.