In lignocellulose-to-hydrogen bioconversion, reducing the concentration of chemical agents in pretreatment is of great interest. In this study, rice straw (RS) pretreated at reduced NaOH and urea (NU) concentrations was evaluated. Results showed that the composition of RS exhibited excellent pretreatment performance at a reduced concentration of NU. When the concentration of NaOH was decreased to 3 wt% in combination with 6 wt% urea, the lignin was reduced by 59.52% with a cellulose and hemicellulose loss of less than 17%. Moreover, extending the pretreatment time at a low concentration of NU could effectively promote the biodegradability of RS. Upon fermentation by Thermoanaerobacterium thermosaccharolyticum M18 for H-2 production, the H-2 production increased up to 213.06 mL/g with a substrate treated by 3 wt% NaOH/6 wt% urea at low solid loading for 15 d, which was 16.31% higher than the counterpart subjected to a 7 wt% NaOH/12 wt% urea pretreatment. The present results suggest the NU pretreatment can be carried out at low concentrations to improve the conversion of RS into bio-H-2 production. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.