The effect of different gases, CO(2) concentration, and separation of CO(2) from reaction system on photo-fermentation H(2) production was investigated by batch culture in this study. Experimental results showed that different gases (Ar,N(2),CO(2), and air) as gas phase have obviously affected on photo-H(2) production and a high concentration of CO(2) can inhibit the growth and H(2) evolution of Rhodopseudomonas faecalis RLD-53. When CO(2) concentration at 5%, cell increased most rapidly the specific growth rate of 0.489 g/l/h and the specific growth rate fell to be 0.265 g/l/h when CO(2) concentration at 40%. However, the growth of RLD-53 at CO(2) concentration of 60-100% was almost completely inhibited. At CO(2) concentrations of 5% and 10%, the maximum H(2) yield was 2.54 and 2.59 mol-H(2)/mol acetate, respectively, and it was similar with the control (2.61 mol-H(2)/mol acetate). H(2) not produced when CO(2) concentration at 60-100%. In conclusion, separation of CO(2) from reaction system can stimulate H(2) production in the entire photo-H(2) production process and H(2) yield increased about 12.8-18.85% than the control.