In this paper, the effect of hydraulic retention time (HRT, 16 h-4 h) on fermentative hydrogen production by mixed cultures was firstly investigated in a sucrose-fed anaerobic continuous stirred tank reactor (CSTR) at 35 degrees C and initial pH 8.79. After stable operations at HRT of 16-6 h, the bioreactor became unstable when the HRT was lowered to 4 h. The maximum hydrogen yield reached 3.28 mol H-2/mol-Sucrose at HRT 4 h. Supplementation of Cu2+ at HRT 4 h improved the operation stability through enhancement of substrate degradation efficiency. The effect of Cu2+ concentration ranging from 1.28 to 102.4 mg/L on fermentative hydrogen production was studied. The results showed that Cu2+ was able to enhance the hydrogen production yield with increasing Cu2+ concentration from 1.28 to 6.4 mg/L. The maximum hydrogen yield of 3.31 mol H-2/mol-Sucrose and the maximum hydrogen production rate of 14.44 L H-2/Day/L-Reactor were obtained at 6.4 mg/L Cu2+ and HRT 4 h Cu2+ at much higher concentration could inhibit the hydrogen production, but it could increase substrate degradation efficiency (12.8 and 25.6 mg/L.Cu2+). The concentration of Cu2+ had effect on the distribution of soluble metabolite. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.