In diatoms, metabolic activity during long dark periods leads to a chlororespiratory electron flow, which is accompanied by the build-up of a proton gradient strong enough to activate the diadinoxanthin (Ddx) de-epoxidation reaction of the Ddx cycle. In the present study, the impact of chlororespiration on non-photochemical quenching (NPQ) of chlorophyll fluorescence and the regulation of the Ddx cycle in the diatom Thalassiosira pseudonana was investigated by manipulation of the redox state of the photosynthetic electron transport chain during darkness. The response of a transfer of T. pseudonana cells from growth light conditions to 60 min darkness was found to depend on oxygen: in its presence there was no significant reduction of the PQ pool and no de-epoxidation of Ddx to diatoxanthin (Dtx). Under anaerobic conditions a high reduction state of the electron transport chain and a slow but steady de-epoxidation of Ddx was observed, which resulted in a significant accumulation of Dtx after 60 min of anaerobiosis. Unexpectedly, this high concentration of Dtx did not induce a correspondingly high NPQ as it would have been observed with Dtx formed under high light conditions. However, the sensitivity of NPQ to Dtx in cells kept under dark anaerobic conditions increased during reoxygenation and far-red (FR) light illumination. The results are discussed with respect to the activation of the de-epoxidation reaction and the formation of NPQ and their dependence on the extent of the proton gradient across the thylakoid membrane.