The cytochrome b 6 f (cytb 6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1,2,3. Electron transfer within cytb 6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH 2 ) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb 6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb 6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb 6 f monomer near the PQ oxidation site (Q p ) adjacent to haem b p and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Q p site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Q n ) on the PQ1 side and committing the electron transfer network to turnover at the occupied Q n site in the neighbouring monomer. A conformational switch involving the haem c n propionate promotes two-electron, two-proton reduction at the Q n site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Q p and Q n sites in opposite monomers during the Q cycle. The spinach cytb 6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.