Many bacterial pathogens utilize a specialized secretion system, termed type III secretion system (T3SS), to translocate effector proteins into host cells and establish bacterial infection. The T3SS is anchored within the bacterial membranes and contains a long needle/filament that extends toward the host-cell and forms, at its distal end, a pore complex within the host membrane. The T3SS pore complex consists of two bacterial proteins, termed SctB and SctE, which have conflicting targeting indications; a signal sequence that targets to secretion to the extracellular environment via the T3SS, and transmembrane domains (TMDs) that target to membrane localization. In this study, we investigate whether the TMD sequences of SctB and SctE have special features that differentiate them from classical TMDs and allow them to escape bacterial membrane integration. For this purpose, we exchanged the SctB and SctE native TMDs for alternative hydrophobic sequences and found that the TMD sequences of SctB and SctE dictate membrane destination (bacterial versus host membrane). Moreover, we examined the role of the SctB TMD sequence in the activity of the full-length protein, post secretion, and found that the TMD does not serve only as a hydrophobic segment, but is also involved in the ability of the protein to translocate itself and other proteins into and across the host cell membrane.