β-Lactoglobulin (β-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured β-LG. In the present study, we show a dramatic increase in β-LG immunoreactivity when heating raw milk between 70 and 80°C. To map out the specific epitope of β-LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66-76) of β-LG. Circular dichroic spectral analysis shows that carboxymethylation on β-LG not only resulted in a substantial loss of β-configuration but also exerted a 10 times increase in immunoreactivity as compared with heated β-LG. The result suggests that a further disordered structure occurred in β-LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated β-LG and its binding to retinol or palmitic acid. Most interestingly, pH 9-10, which neutralizes the Lys groups of β-LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of β-LG participated in the thermal denaturation between 70 and 80°C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.