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Patulin (PAT), mainly produced by <i>Penicillium expansum</i>, is a potential threat to health. In recent years, PAT removal using antagonistic yeasts has become a hot research topic. <i>Meyerozyma guilliermondii</i>, 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 of <i>M. guilliermondii</i> over PAT exposure and its detoxification enzymes are not apparent. In this study, transcriptomics is used to unveil the molecular responses of <i>M. guilliermondii</i> on 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 of <i>M. guilliermondii</i>, which could be helpful to further accelerate the commercial application of antagonistic yeast toward mycotoxin decontamination.