Abstract The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane β barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TspB transporter FhaC. This revealed substantial, spontaneous conformational changes with a portion of the POTRA2 domain coming close to the lipid bilayer and surface loops. Specifically, the amphipathic β hairpin immediately preceding the first barrel strand can insert into the β barrel. We propose that these motions enlarge the channel and may hoist the substrate into it for secretion. An anchor region at the interface of the β barrel and the POTRA2 domain stabilizes the transporter in the course of secretion. Our data propose a solution to the conundrum how these proteins mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.