This study investigated the effect of EDTA concentration in medium on the growth, hydrogen production and nitrogenase activity of Rhodopseudomonas faecalis RLD-53 by batch cultures. Experimental results indicated that bacterial growth and hydrogen production were strongly inhibited with EDTA concentration increasing to 0.6-0.7 g/L. However, the lag time of hydrogen production and the trends of biomass at EDTA concentration of 0-0.5 g/L were similar. The maximum cumulative hydrogen volume of 3325 ml H-2/L culture, hydrogen production rate of 27.6 ml H-2/L/h and hydrogen yield of 2.97 mol H-2/mol acetate were obtained when EDTA concentration was at 0.3 g/L, and the maximum OD660 attained 3.83. And the nitrogenase activity also reached a maximum value of 1331.9 mu l-C2H4/h/mg dry weight in the medium containing Fe2+ and EDTA. These results showed that a proper concentration of EDTA can promote the availability of iron, thereby further enhancing the activity of nitrogenase and photo-hydrogen production. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.