Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI‐2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI‐2‐encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild‐type Salmonella inhibited fusion of Salmonella‐containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome–endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis.