<p>Due to fluctuations in sunlight intensity, balancing light-harvesting efficiency and photoprotection is critical for plant fitness. To achieve this balance, plants have evolved a highly regulated mechanism, non-photochemical quenching (NPQ), which is activated under excess light to dissipate absorbed energy and deactivated under low light to preserve photosynthetic efficiency. A key component that allows NPQ to respond rapidly to changing light conditions is photosystem II subunit S (PsbS). Several hypotheses have been proposed to explain the working mechanism of PsbS, including the idea that it modulates thylakoid membrane macrostructure and protein mobility by altering membrane fluidity. To test this hypothesis, we employed a proteoliposome system containing only thylakoid lipids, PsbS, and the fluorescent probe laurdan, enabling direct detection of PsbS–lipid interactions under different pH conditions. Our results show that the presence of PsbS increases membrane fluidity, but the magnitude of this change in the overall bilayer is small compared with temperature-induced effects. These findings indicate that direct interactions between PsbS and thylakoid lipids are insufficient to account for NPQ regulation and suggest that interactions with other components in the thylakoid membranes are likely essential. This study provides insight into the functional mechanism of PsbS and establishes a framework for further investigation of PsbS-mediated regulation of NPQ and its role in optimizing photosynthetic performance under fluctuating light.</p>