PsbS plays a major role in activating the photoprotection mechanism known as "non-photochemical quenching," which dissipates chlorophyll excited states exceeding the capacity for photosynthetic electron transport. PsbS activity is known to be triggered by low lumenal pH. However, the molecular mechanism by which this subunit regulates light harvesting efficiency is still unknown. Here we show that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins (CP29 and CP24) and the trimeric LHCII-M. Dissociation of this supercomplex is indispensable for the onset of non-photochemical fluorescence quenching in high light, strongly suggesting that protein subunits catalyzing the reaction of heat dissipation are buried into the complex and thus not available for interaction with PsbS. Consistently, we showed that knock-out mutants on two subunits participating to the B4C complex were strongly affected in heat dissipation. Direct observation by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpreted these results to mean that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions.