Hydrogen (H-2) is considered as the most attractive alternative fuel and photosynthetic-H-2 production by green microalgae is regarded as the most promising biofuel. However, incompatibility in the simultaneous O-2 and H-2 evolution from microalgae prevented the H-2 production industry, and the inconvenience of culture medium change for sulfur deprivation treatment also makes it not cost-effective. In this study, a new strategy on regulation of H-2 production by a model microalga Chlamydomonas reinhardtii was investigated by using artificial miRNA (amiRNA) technology. A heat-inducible expression vector containing the amiRNA targeting OEE2 gene (a photosystem II related protein, oxygen evolving enhancer (OEE2)) was constructed and transformed into C. reinhardtii. With a more rapidly O-2 consumption, the transgenic alga showed that OEE2 gene knock down was correlated with the higher H-2 production. The highest H-2 yield was observed after the second heat shock made to the same transgenic algal culture, about 2-fold more than the control under the same condition. With no need to change medium, our results suggested a prospective way for continuous H-2 production by green algae for industry applications. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.