Fomitopsis pinicola is a medicinal fungus in basidiomycetes with a wide range of medicinal properties, including anti-cancer, anti-inflammatory, anti-diabetic, and anti-obesity properties. In this study, the protective effect of F. pinicola mycelia polysaccharides (FPMPS) against alcohol-induced injury was assessed using serum biochemical indices and hepatic and cecal morphology. Moreover, the potential mechanisms of action of FPMPS against acute alcohol-induced liver injury in vivo were investigated using gut microbiota and transcriptome analysis. The result showed that FPMPS modulated alcohol metabolism, restored serum lipid levels, and maintained hepatic and cecal morphology. FPMPS ameliorated gut microbial disorders caused by alcohol to protect the liver. In addition, FPMPS regulated retinol metabolism, bile secretion, inflammatory mediator regulation of TRP channels, and the PI3K-Akt signaling pathway to relieve acute alcoholic liver injury. In addition, the retinol metabolic pathway plays an important role in the preventive effects of FPMPS against alcoholic liver injury. Therefore, FPMPS can be used as a potential new drug for the prevention of acute alcohol-induced liver injury and as a health food with auxiliary functions.