ABSTRACTIntegrative conjugative elements (ICEs) are mobile blocks of DNA that can contribute to bacterial evolution by self-directed transmission of advantageous traits. Here, we analyze the activity of a putative 65-kb ICE harbored byLegionella pneumophilausing molecular genetics, conjugation assays, a phenotype microarray screen, and macrophage infections. The element transferred to a naiveL. pneumophilastrain, integrated site-specifically, and conferred increased resistance to oxacillin, penicillin, hydrogen peroxide, and bleach. Furthermore, the element increased survival ofL. pneumophilawithin restrictive mouse macrophages. In particular, this ICE protectsL. pneumophilafrom phagocyte oxidase activity, since mutation of the macrophage NADPH oxidase eliminated the fitness difference between strains that carried and those that lacked the mobile element. Renamed ICE-βox (forβ-lactam antibiotics andoxidative stress), this transposable element is predicted to contribute to the emergence ofL. pneumophilastrains that are more fit in natural and engineered water systems and in macrophages.IMPORTANCEBacteria evolve rapidly by acquiring new traits via horizontal gene transfer. Integrative conjugative elements (ICEs) are mobile blocks of DNA that encode the machinery necessary to spread among bacterial populations. ICEs transfer antibiotic resistance and other bacterial survival factors as cargo genes carried within the element. Here, we show thatLegionella pneumophila, the causative agent of Legionnaires’ disease, carries ICE-βox, which enhances the resistance of this opportunistic pathogen to bleach and β-lactam antibiotics. Moreover,L. pneumophilastrains encoding ICE-βox are more resistant to macrophages that carry phagocyte oxidase. Accordingly, ICE-βox is predicted to increase the fitness ofL. pneumophilain natural and engineered waters and in humans. To our knowledge, this is the first description of an ICE that confers oxidative stress resistance to a nosocomial pathogen.