Abstract Macrophages provide a first line of defense against microorganisms, and while some mechanisms to kill pathogens such as the oxidative burst are well described, others are still undefined or unknown. Here, we report that the Rab32 guanosine triphosphatase and its guanine nucleotide exchange factor BLOC-3 (biogenesis of lysosome-related organelles complex–3) are central components of a trafficking pathway that controls both bacterial and fungal intracellular pathogens. This host-defense mechanism is active in both human and murine macrophages and is independent of well-known antimicrobial mechanisms such as the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)–dependent oxidative burst, production of nitric oxide, and antimicrobial peptides. To survive in human macrophages, Salmonella Typhi actively counteracts the Rab32/BLOC-3 pathway through its Salmonella pathogenicity island-1–encoded type III secretion system. These findings demonstrate that the Rab32/BLOC-3 pathway is a novel and universal host-defense pathway and protects mammalian species from various pathogens. INTRODUCTION Cells of our innate immune system, e.g., macrophages, are involved in the first line of defense against microorganisms. After phagocytosis, macrophages can eliminate most of the microorganisms they encounter by directing them in intracellular compartments where conditions are not compatible with microorganism life. A key strategy used by macrophages to kill microbes is the production of reactive oxygen species (ROS) through activation of the NADPH oxidase complex that is assembled on cellular membranes in response to infection ( 1 ). Other mechanisms, such as the production of nitric oxide, or cathelicidin-related antimicrobial peptide (Cramp), can also mediate bacterial killing ( 2 ). […]