Ferredoxin I (FdI), encoded by fdxN gene, is proved to be the main electron donor of nitrogenase for hydrogen production. In this work, fdxN gene overexpression was implemented in a mutant MHY01, which was constructed by inserting fdxN gene into the hupSL region in Rhodobacter sphaeroides HY01 genome. Its photo-fermentative H-2 production performance was studied. The results showed that the expression level of fdxN and nitrogenase activity in MHY01 (hupSL::fdxN) were enhanced by 177% and 61.7% respectively compared with that of wild type HY01. Using 25 mM acetate and 34 mM butyrate as carbon source and 6 mM L-glutamate as nitrogen source, the maximum H-2 production rate was 156.1 mL/(L.h.), which was increased by 50.7% compared with that of HY01. The maximum H-2 production rates of MHY01 were enhanced by 30.0%, 52.5% and 50.7% compared with those obtained from HY01 at the inoculation size of 5%, 10% and 15% respectively. The results suggested that overexpressing fdxN could enhance the nitrogenase activity and H-2 production performance of purple non-sulfur bacteria. The abundancy of ferredoxin I might limit the efficiency of electron transfer flux associated with the biohydrogen production process. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.