Abstract: To investigate the digestion and absorption of wall-broken Ganoderma lucidum spores in the oral and gastrointestinal tracts and the effects on the intestinal environment. In this study, wall-broken Ganoderma lucidum spore (GLS) by mechanical milling, microwave and ultrasonic methods were subjected to in vitro simulation of human oral, gastric and small intestinal digestive systems and dialysis models, followed by in vitro fermentation of the digested substrate. The loss rate, release of polysaccharides and triterpenoids, bioacceptability and dialysis rate of Ganoderma lucidum spore at each stage of digestion as well as the pH changes of the small intestine digested substrate during 0, 6, 12, 24 and 48 h in vitro fermentation were measured. The results showed that the stomach and small intestine were the main digestion sites of Ganoderma lucidum spores, and the average mass loss rate of the broken wall group reached 23.84%, among which the mechanically milled group was more easily digested with a mass loss rate of 29.46%. 97% of the polysaccharides were dissolved in the gastrointestinal fluid, and the bioacceptability of polysaccharides in the mechanically milled group had the highest level of 87.33%. Triterpenes were only minimally soluble in the simulated digestion of the small intestine in the wall-breaking group, averaging about 1.50±0.04 mg/g, and 95.2% of the triterpenes entered the colon with the precipitation. The in vitro fermentation results showed that the pH value of the fermentation broth decreased continuously from 0 to 12 h and reached the end of fermentation at 12 h. Comparing the digestive characteristics of the three types of wall-broken Ganoderma lucidum spores, the quality of the mechanical milling method of wall-broken was considerable, and it was a preferable choice for enterprise scale wall-broken production. In conclusion, wall-breaking was conducive to increasing the release and bioacceptability of the active ingredients of Ganoderma lucidum spores in the human digestive environment, promoting colonic fermentation and acid production, and helping to regulate the human intestinal environment.