Salmonella Typhimurium is an intracellular pathogen that uses the SPI-2 type III secretion system to deliver virulence proteins across the vacuole membrane surrounding intracellular bacteria. This involves a tightly regulated hierarchy of protein secretion controlled by two molecular switches. We found that SPI-2-encoded proteins SsaP and SsaU are involved in the first but not the second secretion switch. We identify key amino acids of the inner membrane protein SsaV that are required to interact with the so-called gatekeeper protein SsaL and show that the dissociation of SsaV-SsaL causes the second switch, leading to delivery of effector proteins. Our results provide insights into the molecular events controlling virulence-associated type III secretion and suggest a broader model describing how the process is regulated.