Type 3 secretion systems use 3.5-megadalton syringe-like, membrane-embedded 'injectisomes', each containing an ~800-Å-long needle complex to connect intracellular compartments of infectious bacteria and hosts. Here we identify requirements for substrate association with, transport through and exit from the injectisome of Salmonella enterica serovar Typhimurium. This guided the design of substrates that become trapped within the secretion path and enabled visualization of injectisomes in action in situ. We used cryo-EM to define the secretion path, providing a structural explanation as to why effector proteins must be unfolded during transport. Furthermore, trapping of a heterologous substrate in the needle prevents secretion of natural bacterial effectors. Together, the data reveal the path of protein secretion across multiple membranes and show that mechanisms rejecting unacceptable substrates can be undermined, and transport of bacterial effectors across an already assembled type 3 secretion system can be inhibited.