Bacterial symbionts are widespread among metazoans and provide a range of beneficial functions. <i>Wolbachia</i>-mediated protection against viral infection has been extensively demonstrated in <i>Drosophila.</i> In mosquitoes that are artificially transinfected with <i>Drosophila melanogaster Wolbachia</i> (wMel), protection from both viral and bacterial infections has been demonstrated. However, no evidence for <i>Wolbachia</i>-mediated antibacterial protection has been demonstrated in <i>Drosophila</i> to date. Here, we show that the route of infection is key for <i>Wolbachia</i>-mediated antibacterial protection. <i>Drosophila melanogaster</i> carrying <i>Wolbachia</i> showed reduced mortality during enteric—but not systemic—infection with the opportunist pathogen <i>Pseudomonas aeruginosa</i>. <i>Wolbachia</i>-mediated protection was more pronounced in male flies and is associated with increased early expression of the antimicrobial peptide <i>Attacin A</i>, and also increased expression of a reactive oxygen species detoxification gene (<i>Gst D8</i>). These results highlight that the route of infection is important for symbiont-mediated protection from infection, that <i>Wolbachia</i> can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms and that these effects are sexually dimorphic. We discuss the importance of using ecologically relevant routes of infection to gain a better understanding of symbiont-mediated protection
