Abstract Bartonella henselae is a zoonotic pathogen that possesses a flea-cat-flea transmission cycle and causes cat scratch disease in humans via cat scratches and bites. In order to establish infection, B. henselae must overcome oxidative stress damage produced by the mammalian host and arthropod vector. B. henselae encodes for putative Fe2+ and Mn2+ transporter SitABCD. In B. henselae, SitAB knockdown increases sensitivity to hydrogen peroxide. We consistently show that SitAB knockdown decreases the ability of B. henselae to survive in both human endothelial cells and cat fleas, thus demonstrating that the SitABCD transporter plays an important role during the B. henselae infection cycle.