Mechanical unfolding of a single domain protein with engineered disulfide bonds in reducing and oxidizing conditions has been probed by single-molecule force spectroscopy. Reduction of the solvent-accessible disulfide bond by the chemical reducing agent DTT resulted in an increase in the protein contour length, and the kinetics of reduction ware measured at the single-molecule level. In contrast a sequestered disulfide bond was not readily reduced by DTT. We also show that the protein folding kinetics are sensitive to the redox environment, and the differential kinetic of the oxidized and reduced domains were simultaneously measured.