Type I secretion system (T1SS) conduits span the envelope of Gram-negative bacteria and enable single step translocation of proteins, directly from bacterial cytosol into extracellular space and without a periplasmic secretion intermediate. Most of the T1SS substrates belong to ‘Repeats-in-Toxin’ (RTX) proteins characteristic of the presence of the C-terminal secretion signal preceded by single or several blocks of calcium-binding tandem RTX motifs with a prototypical consensus sequence GGxGxDxxx. The RTX repeats remain unfolded inside the Ca2+-depleted bacterial cytosol until their release outside of the bacterial cell into Ca2+-rich environment (>1 mM) where bind Ca2+ ions and fold into a β-roll structure. While the proton motive force and the hydrolysis of ATP by the ABC transporter subunit of T1SS are essential for promoting initial movement of the substrate through the conduit, the mechanism underlying translocation of often very large RTX proteins (>>1,000 residues) is unknown. Here we show that Ca2+-dependent folding of the emerging C-terminal RTX repeats outside the cells acts as an efficient intramolecular ratchet during translocation of the Bordetella pertussis adenylate cyclase toxin (CyaA) through the T1SS conduit. Folding of the RTX repeats is initiated by formation of a structurally conserved C-terminal folding nucleus that governs the vectorial folding of the RTX repeats from the carboxy- towards the amino-terminus of the polypeptide. Sequential assembly of RTX repeats into β-roll continuously prevents backsliding of the substrate through the T1SS duct indicating that a passive diffusion (Brownian ratchet) is sufficient to achieve translocation. These results provide new insights into secretion and activity of major virulence factors of important pathogens, such as the whooping cough agent Bordetella pertussis that is re-emerging in the most developed countries.