his study investigates the effects of sodium selenite on the yield, selenium content, and proteomic profile of Pleurotus pulmonarius (P. pulmonarius). Treatment with 2 μg/mL sodium selenite (PP-Se2) resulted in a 17.87% increase in the yield of P. pulmonarius. In contrast, treatment with 10 μg/mL sodium selenate (PP-Se10) caused a 23.58% reduction in yield. The selenium content exhibited a positive correlation with the sodium selenite concentration in the cultivation substrate. Based on data-independent acquisition (DIA) proteomics analysis, it was revealed that PP-Se2 and PP-Se10 contained 141 differential expressed proteins (DEPs) (64 up-regulated and 77 down-regulated) and 129 DEPs (73 up-regulated and 56 down-regulated), respectively. DEPs in PP-Se2 were predominantly involved in carbon metabolism, ribosomes, peroxisomes, and ABC transporters. In contrast, DEPs in PP-Se10 were primarily enriched in the biosynthesis of secondary metabolites and cysteine and methionine metabolism. These results suggest that P. pulmonarius exhibits distinct metabolic response mechanisms to varying levels of selenium treatment, with these pathways potentially being influenced by selenium concentration. These findings help to fill the gap in selenium proteomics research in edible fungi and provide new theoretical insights for the development of selenium-enriched functional foods.