Patulin (PAT), mainly produced byPenicillium expansum, is a potential threat to health. In recent years, PAT removal using antagonistic yeasts has become a hot research topic.Meyerozyma guilliermondii, isolated by our group, produced antagonistic effects against the postharvest diseases of pears and could degrade PAT in vivo or in vitro. However, the molecular responses ofM. guilliermondiiover PAT exposure and its detoxification enzymes are not apparent. In this study, transcriptomics is used to unveil the molecular responses ofM. guilliermondiion PAT exposure and the enzymes involved in PAT degradation. The functional enrichment of differentially expressed genes indicated that the molecular response mainly includes the up-regulated expression of genes related to resistance and drug-resistance, intracellular transport, growth and reproduction, transcription, DNA damage repair, antioxidant stress to avoid cell damage, and PAT detoxification genes such as short-chain dehydrogenase/reductases. This study elucidates the possible molecular responses and PAT detoxification mechanism ofM. guilliermondii, which could be helpful to further accelerate the commercial application of antagonistic yeast toward mycotoxin decontamination.