A newly isolated strain, HY01, was identified to be Rhodobacter sphaeroides by phylogenetic analysis. Some DNA sequence data indicate that it is highly similar to R. sphaeroides ATCC 17029. The effects of initial pH values, nitrogen sources and carbon sources on hydrogen production were studied. The results showed that pH 7.25 is optimum for its hydrogen production. Among the nitrogen sources of glutamic acid, yeast extract, glycine, ethanolamine and L-aspartic acid, the maximum hydrogen production rate of 98.0 +/- 6.2 mL/(Lh) using 10 mM glutamic acid and maximum hydrogen yield of 7012.5 +/- 150 mL/L using 5 mM glutamic acid was obtained. Maximum hydrogen production rates of 148.7 +/- 4.6 mL/(Lh) and 94.8 mL/(Lh) were obtained under 10 klux and 5 klux light intensity using 7 mM glutamic acid as nitrogen source, respectively. Compared with the reported data, it shows high hydrogen production performance and is a good candidate for further study. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.