Type III secretion (T3S) system is deployed by a wide range of pathogens to manipulate host cell response and establish infection. The T3S system is a syringe-like apparatus that spans across the double membrane of bacteria, protruding 50nm-80nm into the extracellular space and connecting with target cell membrane. In Pseudomonas aeruginosa, the proteins PopB and PopD are secreted and found associated with the target eukaryotic cell membrane. These two proteins are believed to form a transmembrane complex or translocon to allow effector protein translocation. Despite its key role in pathogenesis, the assembly mechanism and structure of this critical transmembrane complex remain largely unknown. In this work, combining biophysical techniques and a cell-based assay we demonstrated that PopB assisted the proper insertion of PopD in the membrane, which led to the formation of a functional translocon. Taking advantage of the cell-based assay, we further identified a novel structural arrangement of PopD associated with cell membranes. Collectively, the results of this dissertation provide insights on the membrane interaction of PopD.