Objectives To investigate the effect of zero-valent iron and copper (Fe/Cu) micro-electrolysis on dark fermentative hydrogen production from glucose by a mixed bacterial consortium and the possible mechanisms of increasing hydrogen yield. Results Compared to zero-valent iron and activated carbon (Fe/C) micro-electrolysis, Fe/Cu micro-electrolysis could increase hydrogen yield by 32.2%, hydrogen production potential by 27.1%, and the maximum hydrogen production rate by 62.0%. Meanwhile, the number of ferrous ions released into the liquid phase with Fe/Cu micro-electrolysis was about 27.0% greater than that released by Fe/C micro-electrolysis, because the dispersion of copper on the surface of iron could markedly improve electrochemical corrosion activity. Metabolic analysis revealed that Fe/C micro-electrolysis promoted acetate formation, which may have been responsible for the observed improvement in fermentative hydrogen production. Further investigation indicated that Fe/Cu micro-electrolysis increased the activity of hydrogenases and stimulated the expression of the [FeFe]-hydrogenase gene. Conclusion Fe/Cu micro-electrolysis is better than Fe/C micro-electrolysis or Fe corrosion alone for dark fermentative hydrogen production.