Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome b6f (Cyt b6f) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site (Qp) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt b6f homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Qp in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt b6f in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration.