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The conserved fold of thioredoxin (Trx)-like thiol/disulfide oxidoreductases contains an invariant cis-proline residue (P76 in Escherichia coliTrx) that is essential for Trx function and that is responsible for the folding rate-limiting step. E. coliTrx contains four additional prolines, which are all in the transconformation in the native state. Notably, a recent study revealed that replacement of all four transprolines in Trx by alanines (Trx variant Trx1P) further slowed the rate-limiting step 25-fold, indicating that one or several of the four transprolines accelerate the trans-to-cistransition of P76 in Trx wild-type (wt). Here, we characterized the folding kinetics of Trx variants containing cisP76 and one or several of the natural transprolines of Trx wt with NMR spectroscopy. First, we demonstrate that the isomerization reaction in Trx1P is a pure two-state transition between two distinct tertiary structures, in which all observed NMR resonances changes follow the same first-order kinetics. Moreover, we show that trans-P68 is the critical residue responsible for the faster folding of wt Trx relative to the single-proline (P76) variant Trx1P, as the two-proline variant Trx2P(P76P68) already folds seven times faster than Trx1P. trans-P34 also accelerates trans-to-cisisomerization of P76, albeit to a smaller extent. Overall, the results demonstrate that transprolines can significantly modulate the kinetics of rate-limiting trans-to-cisproline isomerization in protein folding. Finally, we discuss possible mechanisms of acceleration and the potential significance of a protein-internal folding acceleration mechanism for Trx in a living cell.