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Corn stover (CS) is a promising feedstock for producing biomethane, that can replace diminishing fossil fuels. However, the recalcitrant structure of CS resulted in low degradability in anaerobic digestion (AD). Numerous studies investigated the pretreatment of CS before AD, but the insight mechanism of biomethanation enhancement is not fully revealed. Therefore, this study advanced low-temperature urea-hydrothermal pretreatment of CS, and the biomethane production, substance bioconversion, hydrolase activity, and metagenomic analysis were conducted to unravel the intrinsic mechanisms of pretreatment for the enhanced biomethanation. The results showed that the pretreatment improved 11.5% of the specific surface area of CS, providing 111.5% higher total volatile fatty acids and 19.9% higher reducing sugars than the control, potentially enriching more anaerobic microorganisms. As a result, the pretreated CS achieved 19.1% higher biomethane yield, 9.1% higher volatile solid removal rate, and 3 days shorter digestion time. The mass balance and microbial community succession analysis indicated that the pretreatment reinforced the biomethane conversion from carbohydrate, which was attributed to the rapid enrichment of hydrolytic acidification bacteria (g__unclassified_o__Bacteroidales) (33.2%) and mixotrophic archaea (Methanosarcina) (72.3%). Meanwhile, the activity of cellulase and xylanase was enhanced up to 23.7% and 66.7%. Metagenomic analysis revealed that the combined pretreatment of CS promoted methanogenesis by enhancing various CAZymes secretion (such as oligosaccharide-degrading enzymes), and functional genes expression of hydrolytic, acidification and acetate-methane pathways at days 1–5. The study indicated that the combined pretreatment could influence microbial composition and function by changing the physicochemical properties of the CS, thereby improving methanogenic performance. [Display omitted] • Effect of urea-hydrothermal pretreatment of corn stover was investigated on anaerobic digestion. • Microbial community succession rate was enhanced by the combined pretreatment. • The effect of pretreatment on the hydrolases activity and functional genes were examined. • AcEtate-methane pathway was largely activated by the combined pretreatment. [ABSTRACT FROM AUTHOR]