The bioaccessibility and bioavailability of different cadmium (Cd) levels (low: 7.31 mg/kg, medium: 24.20 mg/kg, high: 41.64 mg/kg) in Boletus griseus were evaluated by establishing a bionic digestive system in vitro. The results showed that the bioaccessibility of high Cd level by gastrointestinal digestion was significantly higher than other two levels. Further, colonic digestion significantly increased the bioaccessibilities of low Cd level (p < 0.05). After intestinal flora fermentation, the bioaccessibilities of different Cd levels significantly decreased (p < 0.05), and high and medium Cd levels had no significant difference (p > 0.05). A Caco-2 monolayer cell model was established to evaluate the bioavailability of Cd. The bioavailabilities of low and high Cd levels by gastrointestinal digestion were 8.75 and 10.58%, and the bioavailabilities increased by 38.17% and 5.20% after colonic digestion, respectively. Furthermore, Cd could affect diversity, composition, and balance of intestinal flora, and the relative abundances of several genera were correlation with Cd levels in B. griseus.