In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase and a response regulator. In Salmonella, the EnvZ/OmpR system responds to osmotic stress and also positively regulates the Salmonella Pathogenicity Island 2 (SPI-2)-encoded type III secretion system required for its survival in the macrophage vacuole. We recently discovered that the EnvZ histidine kinase responds to cytoplasmic osmotic stress and it is capable of sensing osmolality and activating OmpR without being in the membrane. A major question remains as to how Salmonella survive the acidified vacuole and how it responds to cytoplasmic osmotic stress. We reasoned that the Salmonella cytoplasm might acidify upon osmotic stress and during infection to activate OmpR-dependent transcription of the SPI-2 genes. To address these questions, we employed a FRET-based biosensor (“I-switch”) to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion. Our results indicate that the Salmonella cytoplasm acidifies in response to both osmotic stress and vacuolar acidity in an EnvZ/OmpR dependent manner. Microarray analysis and additional experiments confirmed that OmpR represses the cadC/BA operon, which is involved in the recovery from acid stress, thus enabling Salmonella to acidify its cytoplasm. Acidification is required for the secretion of virulence factors; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Our work challenges existing views that bacteria regulate their pH to maintain neutrality, and provides a new model for Salmonella virulence factor secretion and infection. Supported by the Research Center of Excellence in Mechanobiology from the Ministry of Education, Singapore and VA 5I01BX000372 to LJK.