Lentinus sajor-caju has shown potential for the bioconversion of lignocellulosic biomass in various substrates. Here, we evaluated whether L. sajor-caju affects the lignocellulosic biomass, in vitro fermentation and antioxidant properties of Astragalus membranaceus var. mongholicus (AMM) stems. The lignocellulosic biomass content and the antioxidant activity of AMM stems were determined by scanning electron microscopy, chemical component analysis, in vitro fermentation and LC-MS metabolomics after 30 days of fermentation by L. sajor-caju at 25 °C. L. sajor-caju significantly altered the rigid structure of the stems. Compared with the control condition, the lignocellulose contents were significantly reduced (p < 0.001) and improved in vitro digestibility and total volatile fatty acids. In total, 624 differential metabolites, including 201 up-regulated and 423 down-regulated, were identified in unfermented and fermented comparison groups. Correlation analysis indicated that there were strongly correlations of the total phenolic content and total antioxidant capacity. Meanwhile, the differential metabolites were primarily associated with antioxidant activity, with 4(3H)-quinazolinone, dihydrocarvyl acetate and jaceoside being the most representative. In summary, L. sajor-caju altered the composition of the cell wall of AMM stems, thereby enhancing their antioxidant activity.