Previously, we found that marine Chlorella pyrenoidosa strain IOAC707S could generate hydrogen (H-2) under nitrogen deprivation in seawater medium, without dark incubation, N-2 flushing or addition of protonophore. In this work, the H-2 yield of C. pyrenoidosa was enhanced, and the mechanism of H-2 photoproduction was investigated. It was found that C. pyrenoidosa could produce H-2 under hypoxic conditions, which indicated the hydrogenase of C.pyrenoidosa could tolerate a low concentration of O-2. Nitrogen deprivation could lower the efficiency of photosyntem (PS) II photochemical activity and PSII oxygenic activities, which were favorable for rapid establishment of anoxia and efficient H-2 photoproduction, and thus significantly enhance H-2 photoproduction of C. pyrenoidosa. Preculture in nitrogen-deprived medium could further increase the H-2 yield of C. pyrenoidosa, for its more severe inhibition of the oxygen-evolving complex, electron transport and photochemical efficiency of PSII, and PSII oxygenic activities, which facilitated the establishment of anaerobiosis and activation of hydrogenase. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.