subspecies serovar Typhimurium is an intracellular pathogen that invades and colonizes the intestinal epithelium. Following bacterial invasion, is enclosed within a membrane-bound vacuole known as a -containing vacuole (SCV). However, a subset of has the capability to prematurely rupture the SCV and escape, resulting in hyper-replication within the cytosol of epithelial cells. A recently published RNA-seq study provides an overview of cytosolic and vacuolar upregulated genes and highlights vacuolar upregulation. Here, using transcription kinetics, protein production profile, and immunofluorescence microscopy, we showed that PagN is exclusively produced by in SCV. Gentamicin protection and chloroquine resistance assays were performed to demonstrate that deletion of affects replication by affecting the cytosolic bacterial population. This study presents the first example of a virulence factor expressed within the endocytic compartment, which has a significant impact on the dynamics of cytosolic hyper-replication.