The proteomic and lysine acetylation (Kac) changes, accompanying degradation of patulin in Rhodotorula mucilaginosa were analyzed using tandem mass tagging and N6-acetyllysine affinity enrichment followed by LC-MS/MS. Proteomic results showed that expression level of short-chain reductase protein and glutathione S-transferase involved in detoxification was significantly up-regulated. In addition, the expression levels of zinc-binding oxidoreductase and quinone oxidoreductase that are involved in antioxidant process, ABC transport and MFS transport responsible for chemical transport were activated when treated with patulin. The quantitative real time PCR (qRT-PCR) result also indicated these genes expression levels were increased when treated with patulin. Kac changes accompanying degradation of patulin in R. mucilaginosa were also observed. Totally, 130 Kac sites in 103 proteins were differentially expressed under patulin stress. The differentially up expressed modified proteins were mainly involved in tricarboxylic acid cycle and nuclear acid biosynthesis. The differentially down expressed Kac proteins were mainly classified to ribosome, oxidative phosphorylation, protein synthesis and defense to stress process. Our results suggest that patulin exposure prompt R. mucilaginosa to produce a series of actions to resist or degrade patulin, including Kac. In addition, the Kac information in R. mucilaginosa and Kac in response to patulin stress was firstly revealed.