In this work, the corn stover (CS) with concentration of 3%, 6% and 9% (w/v) were pretreated by rumen uid and then used for batched mesophilic biogas production. The results showed that after a 6-day pretreatment, VFAs production of 3.78, 8.27, and 10.4 g/L could be found in 3%, 6% and 9%, respectively. When concerning with biogas production, the highest accumulative methane production of 149.1 mL CH 4 /g VS was achieved by 6% pretreated CS, which was 22% and 45% higher than 3% and 9%, respectively. Also, it was 3.6 times higher than the same concentration of unpretreated CS. The results of microbial community structure revealed highest relative abundance of Firmicutes (45%) and Euryarchaeota (3.9%) in 6% CS, which was bene cial to its high VFAs and methane production. These ndings provide scienti c reference for the utilization of CS and support the development of agricultural waste resource utilization and environmental protection.