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Highlights • Combined ultrasound-acid pretreatment improved H 2 yield of grass by 311.7%. • The combined pretreatment improved the substrate utilization efficiency. • The combined pretreatment induced a more efficient fermentation pathway. • More enrichment of Clostridium contributed to the improvement in H 2 production. Abstract In this study, the dilute acid pretreatment combined with ultrasound was applied to improve fermentative hydrogen production from grass. The experimental results indicated that SCOD and soluble carbohydrate contents of grass was improved by 98.6% and 236.9% after the combined treatment, respectively. Surface morphology (SEM and AFM) and crystallinity analysis revealed that the combined pretreatment process could effectively destroyed the biomass structure and increased their surface area. Owing to the increased soluble organics proportion and better enzymatic accessibility of residual solids, the hydrogen yield reached 42.2 mL/g-dry grass after the combined treatment, which was 311.7%, 190.0% and 35.0% higher in comparison with the control, individual ultrasound and acid pretreated groups, respectively. Meanwhile, the combined treatment also increased the substrate utilization efficiency and induced a more efficient fermentation pathway. Bacterial community analysis revealed that more enrichment of Clostridium and less enrichment of Enterococcus contributed to the improved hydrogen production. [ABSTRACT FROM AUTHOR]