Background: as a plasto quinol oxidase involved in plastoquinol oxidation in higher plants and microalgae, the plastid terminal oxidase (PTOX) was first recognized in the tomato mutant GHOST (GH) and Arabidopsis mutant IMMUTANS (IM). Genome sequence analysis revealed that duplication of the PTOX gene occurs in certain eukaryotic microalgae, but not in cyanobacteria and most higher plants. PTOX may also be involved in carotenoid synthesis and play a critical protective role against stress, such as high light, heat shock and hyperosmosis. However, the connections of PTOX with astaxanthin and bio-hydrogen production and their functional relationship between two PTOX genes in the model green microalga Chlamydomonas is unknown. Results: we successfully knocked down two ptoxs through RNAi in Chlamydomonas, respectively. We demonstrated that expression levels of both PTOXs were increased under stress conditions, and interestingly when one PTOX was silenced the other’s transcriptional level was significantly raised. Conclusions: this shows a complementary relationship under high light condition. In addition, the astaxanthin accumulation level was up-regulated in silenced ptox2 strain, compared to the wide type strain. What’s more, significantly increased hydrogen production was observed in silenced ptox1 strain. In conclusion, PTOXs in Chlamydomonas are connected with not only astaxanthin accumulation but also hydrogen production, and their knock-down strains provide new insights in manipulating microalgae for high light stress tolerant strains, carotenoid production and even biofuels.