Objective: This study investigated the impact of Aspergillus niger lysing polysaccharide monooxygenase (AnLPMO) on in vitro rumen microbial fermentation of rice straw. Methods: AnLPMO was heterologously expressed in Escherichia coli. Fourier transform infrared spectrometry and X-ray photoelectron spectroscopy analyzed the surface structure of rice straw after AnLPMO treatment. Two in vitro experiments, coupled with 16S high-throughput sequencing and quantitative real-time polymerase chain reaction techniques, assessed the influence of AnLPMO on rumen microbial fermentation of rice straw. Results: AnLPMO exhibited peak activity at 40℃ and pH 6.5, with a preference for rice straw xylan hydrolysis, followed by Avicel. AnLPMO application led to the fractional removal of cellulose and hemicelluloses and a notable reduction in the levels of carbon elements and C-C groups present on the surface of rice straw. Compared to the control (no AnLPMO), supplementing AnLPMO at 1.1 to 2.0 U significantly enhanced in vitro digestibility of dry matter (IVDMD, p<0.01), total gas production (p<0.01), and concentrations of total volatile fatty acids (VFA, p<0.01), acetate (p<0.01), and ammonia-N (p<0.01). Particularly, the 1.4 U AnLPMO group showed a 14.8% increase in IVDMD. In the second experiment, compared to deactivated AnLPMO (1.4 U), supplementing bioactive AnLPMO at 1.4 U increased IVDMD (p = 0.01), total gas production (p = 0.04), and concentrations of total VFA (p<0.01), propionate (p<0.01), and ammonia-N (p<0.01), with a limited 9.6% increase in IVDMD. Supplementing AnLPMO stimulated the growth of ruminal bacterial taxa facilitating fiber degradation, including Proteobacteria, Spirochaetes, Succinivibrio, Rikenellaceae_RC9_Gut_Group, Prevotelaceae_UCG-003, Desulfovibrio, Fibrobacter succinogenes, Ruminococcus albus, R. flavefaciens, Prevotella bryantii, P. ruminicola, and Treponema bryantii. Conclusion: These findings highlight AnLPMO's potential as a feed additive for improving rice straw utilization in ruminant production.