Biomass pretreatments represent a necessary route to overcome the natural physico-chemical barriers of recalcitrant feedstocks. However, current biomass pretreatments generally result in generation of various inhibitors (such as furfural derived from pentose), which could inhibit cell growth and decrease biofuel productivity. This study aims to understand the impact of furfural on hydrogen-producing Enterobacter aerogenes in dark fermentation of glucose. When adding 5 mM furfural in fermentation, hydrogen yield unexpectedly increased to 193.7 mL/g compared to 163.5 mL/g (in the absence of furfural); and the associated peak hydrogen production rate increased by 126%. This phenomenon from a thermodynamic perspective was due to the fact that furfural at a low concentration contributes to hydrogen production. A higher concentration of furfural (30 mM) significantly decreased hydrogen yield to 109.1 mL/g owing to severe cell membrane damage. The indicator of half-maximal inhibitory concentration was calculated as 32.5 mM. A postulated metabolic response of E. aerogenes to furfural is that the degradation of low concentrations of furfural (5 mM) involved a reduction reaction of furfural to hydrogen and furfuryl alcohol. However, a high concentration of furfural (30 mM) caused significant cell disfunction in normal metabolism, causing increased deformation degree in bacterial surface.