Summary: The Legionella pneumophilaDot/Icm T4SS injects ~300 protein effector proteins into host cells. Dot/Icm substrates have been proposed to contain a carboxy-terminal signal sequence that is necessary and sufficient for export, although both traits have been demonstrated for only a small fraction of these proteins. In this study, we discovered that export of the substrate SidJ is mediated by dual signal sequences that include a conventional C-terminal domain and a novel internal motif. The C-terminal signal sequence facilitates secretion of SidJ into host cells at early points of infection, whereas the internal signal sequence mediates secretion at later time points. Interestingly, only the internal signal sequence is necessary for complementation of the intracellular growth defect of a ΔsidJ mutant. Although this is the first report of a Dot/Icm substrate being secreted by an internal signal sequence, many other substrates may be exported in a similar manner. In addition, efficient translocation of SidJ is dependent on the chaperone-like type IV adaptors IcmS/IcmW. Five IcmS/IcmW binding domains that are distinct from both signal sequences were elucidated and, interestingly, only secretion mediated by the internal signal sequence requires IcmS/IcmW. Thus, Legionella employs multiple sophisticated molecular mechanisms to regulate the export of SidJ. Export of the protein SidJ, a Legionella Dot/Icm type IV secretion substrate, is mediated by a c-terminal and internal signal sequence that share homology. In addition, optimal secretion of SidJ requires multiple binding sites for the chaperone IcmS/IcmW.