The shiitake mushroom is the most commonly cultivated edible mushroom in the world, and is rich in protein. This study aims to obtain the peptides with α-glucosidase inhibition activity from shiitake mushroom protein hydrolysate. The conditions of enzymatic hydrolysis of shiitake mushroom protein were optimized by response surface test. The results showed that the optimal conditions were as follows: the E/S was 3390 U/g, the solid–liquid ratio was 1:20, the hydrolysis temperature and time were 46 °C and 3.4 h, respectively, and the pH was 7. The active peptides were separated by gel filtration and identified by LC-MS/MS analysis and virtual screening. The results indicated that fourteen peptides were identified by LC-MS/MS. Among them, four new peptides (EGEPKLP, KDDLRSP, TPELKL, and LDYGKL) with the higher docking score were selected and chemically synthesized to verify their inhibition activity. The IC50 values of EGEPKLP, KDDLRSP, TPELKL, and LDYGKL for α-glucosidase inhibition activity ranged from 452 ± 36 μmol/L to 696 ± 39 μmol/L. The molecular docking results showed that the hydrogen bond and arene–cation bond were the two major interactions between four peptides and 2QMJ. The hydrogen bonds were crucial to the inhibition activity of α-glucosidase. The results indicate the potential of using the peptides from shiitake mushroom protein as functional food with α-glucosidase inhibition activity.