Aspirin, one of most famous old drugs, can exert its functions through non-covalent inhibition and covalent acetylation (N-acetylation and O-acetylation) of protein targets. Here we identified lysine salicylation (Ksa) as a novel mechanism of action of aspirin in cell lines and mice. Unexpectedly, we found that aspirin treatment resulted in a reduction in glycolysis, in which the conversion of fructose-1,6-bisphosphate (FBP) to glyceraldehyde 3-phosphate (GAP) was significantly inhibited. Mechanistically, the deceased FBP turnover was unrelated or partially related to the changes in ALDOA itself or its acetylation. Instead, aspirin treatment increased cellular salicyl-CoA which salicylated ALDOA at Lys13 and Lys42, leading to reduced ALDOA activity and FBP to GAP transformation. Collectively, the discovery of a new mode of action for aspirin may expand its applications in the biomedical field.