Ganoderma lucidum, one of the best-known medicinal mushrooms in the world, produces more than 400 different bioactive compounds. By the combination of affinity enrichment and high-resolution LC-MS/MS analysis, large-scale lysine succinylome analysis was performed. Altogether, 742 lysine succinylation sites in 382 protein groups were identified. Bioinformatics analysis revealed that the modified proteins were involved in multiple cellular functions and participated in a wide variety of biological processes such as carbon metabolism and biosynthesis of secondary metabolites.