The non-photochemical quenching of light energy as heat (NPQ) is an important photoprotective mechanism that is activated in plants when light absorption exceeds the capacity of light utilization in photosynthesis. The PsbS protein plays a central role in this process and is supposed to activate NPQ through specific, light-regulated interactions with photosystem (PS) II antenna proteins. However, NPQ-specific interaction partners of PsbS in the thylakoid membrane are still unknown. Here, we have determined the localization and protein interactions of PsbS in thylakoid membranes in the NPQ-inactive (dark) and NPQ-active (light) states. Our results corroborate a localization of PsbS in PSII supercomplexes and support the model that the light activation of NPQ is based on the monomerization of dimeric PsbS and a light-induced enhanced interaction of PsbS with Lhcb1, the major component of trimeric light-harvesting complexes in PSII.