The type VI secretion system (T6SS) of Salmonella enterica serovar Typhi (S. typhi) is associated with Salmonella pathogenicity island 6 (SPI-6). Though the T6SS gene cluster is intact in S. typhi, the protein complex is believed to be non-functional due to the presence of a pseudogene form of SciI (VipB homolog), a key component. We detected the SciK-his(6) in the supernatant of the wild type strain of S. typhi containing the plasmid over-expressing SciK (hcp homolog) with a his(6) epitope at the C-terminus, which suggested that the T6SS in S. typhi is functional. We also identified four genes that were essential to T6SS function: sciC (vasA homolog), sciS (vasK homolog), sciG (clpV homolog), and vrgS (vgrG homolog). Further analysis revealed that S. typhi T6SS is cytotoxic to human epithelial cells, but does not influence bacterial growth and mobility. RcsB, PmrA, and Hfq were identified as regulators of S. typhi T6SS gene expression; however, PhoP appears to not be involved. Taken together, the data demonstrate the functionality of S. typhi T6SS and confirm the important role of T6SS for S. typhi's ability to invade and infect epithelial cells.