The physiological osmolyte trimethylamine-N-oxide (TMAO) stabilizes proteins by decreasing the entropy of the unfolded state through a solvophobic effect. Our studies on the effect of TMAO on the reductive unfolding of onconase (ONC) to form its reductive intermediate, des [30-75], indicate that TMAO diminishes the reductive unfolding rate of the protein although it does not significantly affect the stability of the native protein relative to its denatured state. Since the reductive unfolding of ONC is a local event, our studies provide direct evidence for a TMAO-induced local structural change that reduces the rate of redox-dependent protein unfolding. The implications of our findings for protein folding/unfolding are discussed.