Abstract: To further advance the product development of different grades of Lithocarpus litseifolius (Hance) Chun, this study used premium-grade Lithocarpus litseifolius (Hance) Chun extract (PLE) and first-grade Lithocarpus litseifolius (Hance) Chun extract (FLE) as research subjects. An in vitro oral-gastric-intestinal digestion and colonic fermentation model was established to evaluate the simulated digestion and fermentation characteristics, while investigating their impact on the gut microbiota. The results showed that both PLE and FLE were rich in functional active components, including trilobatin, phloridzin, and gallocatechin (GC). Trilobatin, the most abundant monomeric component, was present at 13.87% in PLE and 12.15% in FLE. Following simulated digestion, its retention rates were 49.89% and 37.28% for PLE and FLE, respectively, indicating that PLE exhibited greater stability during digestion. Bio-activity evaluation revealed that PLE demonstrated significantly higher DPPH radical scavenging activity (529.18 μg Vc TE/mg), α-amylase inhibitory activity (322.03 μg Acb TE/mg), and pancreatic lipase inhibitory activity (91.19 μg Orlistat TE/mg) compared to FLE (315.78, 207.87, and 77.72 μg TE/mg, respectively), suggesting stronger antioxidant, hypoglycemic, and lipid-lowering potentials. Although all activities decreased after simulated digestion, they were partially retained. Notably, the retention rates of those activities for PLE were 58.81%, 58.33%, and 45.51%, respectively, which were also higher than those for FLE (40.91%, 47.01%, and 32.26%), indicating that PLE maintained better residual activity after digestion. After 24 h of simulated fermentation, both extracts significantly promoted the synthesis of short-chain fatty acids (SCFAs) and lowered the pH of the system. Notably, PLE exhibited a stronger promotion effect on SCFAs production compared to FLE. Furthermore, both extracts modulated the gut microbiota composition, with PLE demonstrating a more pronounced effect in increasing the abundance of beneficial bacteria such as Bifidobacterium and inhibiting opportunistic pathogens including Proteus and Citrobacter, thereby more effectively maintaining gut homeostasis. In conclusion, Lithocarpus litseifolius (Hance) Chun extract was a functional ingredient with potential health benefits. Furthermore, PLE is superior to FLE in terms of active component content, digestive stability, functional activity retention, and prebiotic potential during colonic fermentation, demonstrating greater application potential in functional food development.