3-phosphorylated phosphoinositides (3-PtdIns) orchestrate endocytic trafficking pathways exploited by intracellular pathogens such as Salmonella to gain entry into the cell. To infect the host, Salmonellae subvert its normal macropinocytic activity, manipulating the process to generate an intracellular replicative niche. Disruption of the PtdIns(5) kinase, PIKfyve, be it by interfering mutant, siRNA-mediated knockdown or pharmacological means, inhibits the intracellular replication of Salmonella enterica serovar typhimurium in epithelial cells. Monitoring the dynamics of macropinocytosis by time-lapse 3D (4D) videomicroscopy revealed a new and essential role for PI(3,5)P(2) in macropinosome-late endosome/lysosome fusion, which is distinct from that of the small GTPase Rab7. This PI(3,5)P(2)-dependent step is required for the proper maturation of the Salmonella-containing vacuole (SCV) through the formation of Salmonella-induced filaments (SIFs) and for the engagement of the Salmonella pathogenicity island 2-encoded type 3 secretion system (SPI2-T3SS). Finally, although inhibition of PIKfyve in macrophages did inhibit Salmonella replication, it also appears to disrupt the macrophage's bactericidal response.