Background: Sanghuangporus sanghuang is a well-known traditional medicinal mushroom associated with mulberry, which has enormous economic and medical value. Despite being known for many years, the regulatory mechanisms of bioactive compounds biosynthesis in this medicinal mushroom are still unclear.Lysine malonylation is one of a post-translational modifications that has many critical functions in various aspects of cell metabolism. However, at present we do not know its role in S. sanghuang. In this study, a global investigation of lysine malonylome in S. sanghuang was therefore carried out. Results: In total, 714 malonyl modification sites were matched to 255 different proteins. The analysis indicated that malonyl modifications were involved in a wide range of acellular functions and displayed a distinct subcellular localization. Bioinformatics analysis indicated that malonylated proteins were engaged in different metabolic pathways, including glyoxylate and dicarboxylate metabolism, glycolysis/gluconeogenesis, and the tricarboxylic acid (TCA) cycle. Notably, a total of 26 enzymes related to triterpene and polysaccharide biosynthesis were found to be malonylated, indicating an indispensable role of lysine malonylation in bioactive compounds biosynthesis in S. sanghuang. Conclusions: These findings suggest that malonylation is associated with many metabolic pathways, particularly the metabolism of bioactive compounds triterpene and polysaccharide. This paper represents the first comprehensive survey of malonylation in S. sanghuang and provides an important data for further study on the physiological function of lysine malonylation in S. sanghuang and other medicinal mushrooms.