Screening new Clostridium strains that can efficiently utilize lignocellulose to produce hydrogen is extremely important for dark fermentative hydrogen production. In this study, a mesophilic hydrogen-producing bacterium, identified as Clostridium populeti FZ10, was newly isolated from compost acclimated by microcrystalline cellulose. The strain could produce hydrogen from various cellulosic substrates. The performances of hydrogen production from microcrystalline cellulose (MCC) and corn stalk (CS) were especially investigated. The maximum hydrogen yield and hydrogen production rate from MCC were 177.5 ± 4.8 mL/g and 7.7 ± 0.2 mL·g−1·h−1, respectively. Furthermore, scanning electron microscopy (SEM) images showed that the structure of CS was destroyed after fermentation, which could be attributed to the presence of exoglucanase, endoglucanase, β-glucosidase and xylanase produced by Clostridium populeti FZ10. The maximum hydrogen yield and hydrogen production rate from CS were 92.5 ± 3.7 mL/g and 5.9 ± 0.2 mL·g−1·h−1,respectively, with a cellulose degradation of 47.2 ± 2.3% and a hemicellulose degradation of 58.1 ± 2.0%. This study demonstrates that Clostridium populeti FZ10 is an ideal candidate for directly converting lignocellulose into biohydrogen under mesophilic conditions. The discovery of strain C. populeti FZ10 has special significance in the field of bioenergy.