The efficient translocation of the bacterial toxin adenylate cyclase toxin (CyaA) from the bacterial cytosol to the extracellular environment by the type 1 secretion system (T1SS) is essential for the toxin to function. To understand the molecular features that are responsible for the efficient translocation of CyaA, here we used optical tweezers to investigate the mechanical properties and conformational dynamics of the RTX domain of CyaA at the single molecule level. Our results revealed that apo-RTX behaves like an ideal random coil. This property allows the T1SS to translocate RTX without overcoming the enthalpic resistance. In contrast, the folded holo-RTX is mechancially stable, and its folding occurs in a vectorial, cotranslocational fashion starting from its C-terminus. Moreover, our results showed that the folding of holo-RTX generates a stretching force, which can further facilitate the translocation of RTX. Our results highlight the important role played by the Ca2+-triggered folding of RTX in the translocation of RTX and provide mechanistic insights into the mechanical design that governs the efficient translocation of RTX.