A new isolated photosynthetic bacterium, Rubrivivax gelatinosus M002, can produce hydrogen with glucose or lactate as sole carbon source, and grow on butyrate and acetate without hydrogen evolution. Experiments on studying its hydrogen production performance from glucose mixed with acetate, butyrate or lactate were carried out. The results showed that the hydrogen yield increased significantly and the pH value of the photo-fermentations could retain around 7 in these mixed carbon sources cultures. A hydrogen yield of 9.9 mol H-2/mol-glucose was observed when 20 mM acetate and 15 mM glucose was co-fed as substrate. The maximum hydrogen production rate was 44 mL/(L.h), which was 37.5% higher than the highest rate obtained with glucose as sole carbon source. The results suggest an alternative way for high-yield hydrogen production with mixed carbon source in one-step process instead of two-step fermentation process. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.