The effects of L-cysteine concentration on biohydrogen production by Enterobacterium Bacterium M580 were investigated in batch cultivation. The experimental results showed that L-cysteine could enhance the cell growth, hydrogen production rate and hydrogen yield when its concentration was less than 500 mg-L-1, while it had negative effects when its concentration was higher than 500 mg-L-I. The hydrogen production was the highest [1.29 mol-mol(-1) (H-2/glucose)] when 300 mg-L-1 L-cysteine was added into the culture, and the yield was 9.4% higher than that in the control. The oxidation-reduction potential (ORP), which was influenced by L-cysteine, also affected hydrogen production. The ORP values were in the range -300 mV to -150 mV when the L-cysteine concentration was higher than 500 mg-L-1. Although the ORP in this range was favorable for hydrogen production, it was not suitable for the biomass growth. Hence, less hydrogen was produced. When the L-cysteine concentration was lower than 500 mg-L-I, the ORP was more suitable for both biomass growth and hydrogen production. In addition, at least 91% glucose was consumed when L-cysteine was added to the culture media, compared to the 97.37% consumption without L-cysteine added.