The effects of Fe2+, Mg2+ ions and L-cysteine on hydrogen production and cell growth of Clostridium beijerinckii RZF-1108 were investigated, and [FeFe]-hydrogenase (hydA) gene expression under different conditions was evaluated using quantitative reverse transcription PCR (qRT-PCR). Best concentration of Fe2+ stimulated hydrogen production, cell biomass and hydA gene expression in C. beijerinckii RZF-1108. Mg2+ increased cell growth and hydrogen evolution, but negatively facilitated hydrogen production rate and hydA gene expression. L-cysteine slightly stimulated hydA gene expression, but not hydrogen production in strain RZF-1108. A maximum hydrogen yield of 1.96 mol H-2/mol glucose and production rate of 106.0 ml H-2/l medium.h(-1) were obtained using optimized culture medium supplemented with 0.2 g/l FeSO4 center dot 7H(2)O and 0.1 g/l MgCl2 center dot 6H(2)O. These experimental results suggested that the effects of metal ions on hydrogen production by C. beijerinckii RZF-1108 was complicated. Hydrogen production was highly dependent on the cooperation of cell growth and hydrogenase gene expression. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.