Glutamic acid is a flavorful amino acid in Pleurotus eryngii. To explore the effect of high carbon dioxide and low oxygen storage on glutamic acid metabolism in Pleurotus eryngii postharvest, samples were stored in a controlled atmosphere using different gas compositions (CA1 (2% O2), CA2 (2% O2 + 10% CO2), CA3 (2% O2 + 30% CO2), CA4 (2% O2 + 50% CO2), and a normal air gas ratio was used as the control (CK, 20.9% O2 + 0.04% CO2). The malondialdehyde content and antioxidant activity of Pleurotus eryngii were studied over time in storage. The results in combination with clustering analysis showed that CA3 (2% O2 + 30% CO2) treatment played an important role in alleviating the senescence of Pleurotus eryngii postharvest. The effects of CA3 treatment on the phenotype, respiration rate, free amino acid content, and taste change, as well as the activities of protease, glutamate synthase (GOGAT), proline dehydrogenase (PRODH), and glutamate dehydrogenase (GDH) in Pleurotus eryngii were analyzed. The results indicated that compared with the control, CA3 treatment decreased the respiration rate and increased the content of total free amino acids and glutamic acid, thereby maintaining the taste characteristics. In addition, CA3 treatment inhibited the activities of protease, GOGAT, PRODH, and GDH. Therefore, storage at 2% O2 + 30% CO2 could maintain the umami characteristics of Pleurotus eryngii postharvest, and this effect is associated with the inhibition of GDH activity.