Neurodegenerative diseases are considered to be among the diseases most threatening to human beings. Increasing evidence shows that antioxidant hydrolysates/peptides with neuroprotective effects may relieve neurodegenerative diseases. However, related research in mushrooms, one of the richest sources of antioxidant hydrolysates/peptides, is in its infancy. Therefore, the in vitro neuroprotective effects of protein hydrolysates from Pleurotus geesteranus were researched in this study. Proteins were extracted from P. geesteranus and then hydrolyzed by simulated gastrointestinal digestion. The neuroprotective effects of the protein hydrolysates were evaluated by H O -injured PC12 cells. The hydrolysates showed a superior antioxidative ability and had a higher abundance of hydrophobic amino acids (e.g., leucine, alanine, and phenylalanine). Neither cytotoxicity nor the increase of ROS in PC12 cells was observed under treatment with the hydrolysates. However, pre-treatment with the hydrolysates in PC12 cells, which were then injured by H O , markedly attenuated ROS generation and enhanced the activities and mRNA expression of the endogenous antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD)), leading to a 26.68% increase in cell viability. The hydrolysates exhibited strong neuroprotective activity in H O -injured PC12 cells, possibly by reducing ROS generation and enhancing the activity of the antioxidant enzymatic system. PRACTICAL APPLICATIONS: Antioxidant hydrolysates with neuroprotection were obtained from Pleurotus geesteranus proteins by simulating gastrointestinal digestion, which exhibited an excellent pre-protective effect in oxidatively damaged PC12 cells. Further study showed that hydrolysates pre-protection may exert antioxidant activities not only as an exogenous antioxidant to scavenge ROS but also as a gene regulator to modulate the endogenous antioxidant enzymes gene expression. These results indicated that the potential of antioxidant peptides, derived from P. geesteranus through gastrointestinal digestion, could serve as a source of bioactive molecules in the prevention, relief or even treatment of neurodegenerative disorders.