Significance Here, a genome-scale CRISPR/Cas9 screen in epithelial cells using Salmonella , a prototypical intracellular pathogen, led to the discovery of a new role for IFN-I signaling. We found that this canonical antiviral signaling pathway controls the subcellular localization, protein content, pH, and protease activity of lysosomes. IFN-I signaling-dependent lysosomal acidification was associated with heightened Salmonella virulence gene expression and escape into the cytosol, and in vivo studies confirmed a role for epithelial IFN-I signaling in promoting Salmonella systemic infection. We propose that IFN-I signaling-mediated control of lysosome function contributes to host responses to diverse intracellular pathogens and viruses. Investigating how cytokine signaling modifies organelle function will deepen understanding of the cell biology of innate defense.