Interaction between beta-lactoglobulin and single-chain lipids, differing for either the length of the aliphatic chain or the molecular properties of the headgroup, was investigated at neutral and acidic pH to determine the impact on the thermal stability of the protein. Differential scanning calorimetry results with different fatty acids (from C10:0 to C18:0) show a correlation of both melting temperature and unfolding enthalpy of the protein with the ligand binding affinity, and the maximum effect was found for palmitic acid (PLM). The influence of the lipid polar head was investigated by comparing PLM with lyso-palmitoylphosphatidylcholine (LPC), which possesses the same aliphatic chain. At neutral pH, the stabilizing effect of LPC is less favorable compared to PLM. However, fluorescence results revealed that LPC can bind into the protein calyx even at acidic pH, at variance with fatty acids. Molecular dynamics simulations indicated that this difference is due to the ability of the polar head of LPC to interact with the protein loop that regulates the shift (Tanford transition) between open and closed state of the binding site of beta-lactoglobulin. The results provide a rationale for how a ligand has the ability to access the protein active site at acidic conditions by overcoming the Tanford transition, and they demonstrate that beta-lactoglobulin can deliver ligands with tailored properties of the polar head in a wide pH range.