Peanut shell (PS) was the first-time used as a supplementary substrate for fermentative hydrogen production, and the effects of initial substrate concentration, pH and inoculum proportion were investigated with a novel hydrogen producer of Clostridium guangxiense ZGM211(T). The cellulose, hemicellulose and lignin contents of PS were 46.1 +/- 0.9%, 5.6 +/- 0.4% and 27.8 +/- 0.2%, respectively. The hydrogen production with GPS (mixture of glucose and PS) was about 1.7 times more than the sum of respectively with G and PS. Under the optimal fermentation condition of 10 mg/mL glucose with 40 mg/mL PS powder as supplementary substrate, initial pH 6.5 and 4% inoculation proportion, the maximum cumulative hydrogen production of 39.9 mL/g-substrate were obtained. The hydrogen production with GPS was greatly affected by substrate concentration and pH rather than inoculum proportion. PS was found having an buffering effect, and benefit for hydrogen production. The experimental results provide a new idea for the fermentative hydrogen production substrate selection. (C) 2018 Elsevier Ltd. All rights reserved.