Type III secretion (T3S) from enteric bacteria is a co-ordinated process with a hierarchy of secreted proteins. In enteropathogenic and enterohaemorrhagic Escherichia coli, SepL and SepD are essential for translocator but not effector protein export, but how they function to control this differential secretion is not known. This study has focused on the different activities of SepL including membrane localization, SepD binding, EspD export and Tir secretion regulation. Analyses of SepL truncates demonstrated that the different functions associated with SepL can be separated. In particular, SepL with a deletion of 11 amino acids from the C-terminus was able to localize to the bacterial membrane, export translocon proteins but not regulate Tir or other effector protein secretion. From the repertoire of effector proteins only Tir was shown to bind directly to full-length SepL and the C-terminal 48 amino acids of SepL was sufficient to interact with Tir. By synchronizing induction of T3S, it was evident that the Tir-binding capacity of SepL is important to delay the release of effector proteins while the EspADB translocon is secreted. The interaction between Tir and SepL is therefore a critical step that controls the timing of T3S in attaching and effacing pathogens.