Bovine β-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. β-Lactoglobulin A has a free thiol at Cys121, which is buried between the β-barrel and the C-terminal major α-helix. This thiol group was specifically reacted with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCl at pH 7.5, producing a modified β-lactoglobulin (TNB-blg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-blg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional 1H-NMR. The CD spectra of TNB-blg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the α-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-blg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-blg is monomeric while the intact protein is dimeric. In contrast, at pH 2.0, both the intact β-lactoglobulin and TNB-blg were monomeric. The unfolding transition of TNB-blg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The 1H-NMR spectrum for TNB-blg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-blg with dithiothreitol, the intact β-lactoglobulin was regenerated. TNB-blg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.