The effects of overexpressing atpXF genes encoding b(2) subunits of F0F1-ATPase in Rhodobacter sphaeroides HY01 on ATPase, nitrogenase and photo-fermentative hydrogen production activities were studied. The results showed that the expression levels of atpXF and f(1) operon in ZTHBB (F(0)P(+)atpXF(+)) enhanced by 1870% and 40% while its expression levels of f(0) operon and nif decreased by 20% and 50% compared with those of ZTH, respectively; and the expression levels of b(2) subunits, f(0) operon, f(1) operon and nif in ZTHCB (pucP(+)atpXF(+)) enhanced by 370%, 60%, 130% and 160% compared with those of ZTH, respectively. The ATP contents of ZTHBB and ZTHCB increased by 41.5% and 52.8% compared with that of ZTH, and their nitrogenase activities were increased by 30.0% and 41.0% compared with that of ZTH, respectively. When 25 mM acetic acid and 34 mM butyric acid were employed as carbon source and 6 mM L-glutamate was employed as nitrogen source, their hydrogen yields were 7677 176 mL/L (ZTHBB) and 8458 129 mL/L (ZTHCB), which increased by 16.0% and 29.1% compared with that of ZTH, and their maximum hydrogen production rates were 98.0 +/- 4.3 mL/(L.h) (ZTHBB) and 100.1 +/- 5.1 mL/(L.h) (ZTHCB) under the same conditions, which increased by 14.6% and 17.1% compared with that of ZTH, respectively. The results suggest that overexpressing atpXF could enhance the expression level of F0F1-ATPase and hydrogen production performance of purple non-sulfur bacteria. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.