Pheromone-binding protein (PBP) in male moth antennae transports pheromone to the olfactory receptor neuron by undergoing a pH-dependent conformational switch, from PBPB at higher pH to PBPA at lower pH, associated with ligand binding and release, respectively. The characteristic feature of the dramatic protein switch is the pH-dependent reversible coil-helix transition of the C-terminus. In the PBPB conformation at pH >6.0, the C-terminus is exposed to the solvent as a coil while the ligand occupies the hydrophobic pocket. However, in the PBPA conformation at acidic pH, the C-terminus switches to a helix and releases the ligand by outcompeting it for the hydrophobic pocket. In Antheraea polyphemus PBP1 (ApolPBP1), the C-terminus (P129-V142) is composed predominantly of hydrophobic residues except for three strategically located acidic residues: Asp132, Glu137, and Glu141. Here, we report for the first time on the consequences of the mutation of one or more acidic residues in the pH-driven reversible coil-helix transition of the ApolPBP1 C-terminus through biophysical characterization. Mutation of any single acidic residue in the C-terminus to its neutral counterpart destabilizes the helix formation at lower pH; these mutants exist as a mixture of both conformations. However, mutation of the two terminal acidic residues together knocks out the protein switch and adversely affects both ligand binding and release functions. Thus, these mutant proteins remain in the open (PBPB) conformation at all pH levels.