Xylella fastidiosa is a xylem-limited bacterium transmitted by xylem sap-feeding insects (Order Homoptera), being the etiologic agent of serious diseases in a variety of economically important crops, such as the Citrus Variegated Chlorosis (CVC). Infected orange trees produce small and hardened fruits, causing losses of about 100 million dollars a year to the agro-industry of Brazil. The genome of the 9a5c strain of X. fastidiosa, which causes CVC, was completely sequenced enabling large-scale functional studies. In the current work, we report the effects of sublethal concentrations of gomesin, an antimicrobial peptide (AMP) of a spider, on the gene expression profile of X. fastidiosa. AMP are cytotoxic molecules produced by most living organisms, including bacteria and arthropods. Therefore, it is plausible to suppose that X. fastidiosa is exposed to AMP produced by the endophytic microbial community that colonizes citrus xylem and to the AMP of its vectors. Due to the structural characteristics of AMP (the majority is amphipathic and cationic), their main target is the negative membrane of microorganisms. Therefore, studies to determine the mechanisms of action of sublethal concentrations of AMP against bacteria may reveal other microbial targets as well as may point out resistance mechanisms triggered by this kind of treatment. The effects of a sublethal concentration of gomesin on the global gene expression profile of X. fastidiosa were analysed using the DNA-microarray methodology. Our data showed that the majority of differentially expressed genes encode hypothetical proteins. Nonetheless, genes encoding typical stress response molecules, such as chaperones, were up-regulated, indicating that at sublethal concentrations gomesin elicit a stress response-like in X. fastidiosa. Interestingly, the infection of tobacco plants with X. fastidiosa previously exposed to the same sublethal concentration of this AMP reduced the incidence of symptomatic plants to about 44%. This result suggests that in addition to the modulation of the gene expression profile of X. fastidiosa, gomesin may interfere with the interaction between this pathogen and its host.