Spi/Ssa is a unique type three secretion system that functions exclusively when Salmonella enterica is inside eukaryotic cells. Expression of the Spi/Ssa system and its secreted effectors is dependent on SsrB/SpiR, a two-component regulatory system encoded in the SPI-2 pathogenicity island that also harbours the spi/ssa genes. Here we determine that the PhoP/PhoQ two-component system controls the intramacrophage expression of spi/ssa genes by regulating the SsrB/SpiR system. We establish that PhoP regulates transcription of the response regulator SsrB and demonstrate binding of the PhoP protein to the ssrB promoter both in vivo using chromatin immunoprecipitation in Salmonella-infected macrophages, and in vitro using purified PhoP protein. We show that PhoP controls the SpiR sensor post-transcriptionally and identify a region in the 5' untranslated region of the spiR message that is required for this effect. The demonstration that the PhoP/PhoQ system is directly involved in the regulation of the SPI-2 pathogenicity island highlights PhoP/PhoQ's central role in Salmonella virulence. We suggest that different regulatory systems convey distinct signals over time to produce the SsrB/SpiR system, which then modulates expression of the Spi/Ssa apparatus and secreted effectors.