Patulin (PAT) produced by genus of Penicillium spp attracted more and more concern in view of its widespread contamination in food and toxic effects, which has also promoted the research on the reduction of PAT contamination in food. The use of yeast to remove PAT in food is innovative and promising. In this study, we used the yeast Kluyveromyces marxianus XZ1 to degrade PAT, which can remove 90% of PAT (10 μg/mL) within 48 h. XZ1 exhibits high degradation effect on PAT under the conditions of pH range of 3-6, temperatures of 28-37 °C, and initial PAT concentrations below 50 μg/mL. PAT removing by XZ1 was carried out by intracellular enzymes. XZ1 or intracellular enzyme was able to remove 100% of 10 μg/mL PAT in raw apple juice or commercial apple juice within 60 h. Patulin oxidoreductase (KmPAO) was identified as a potential PTA-degrading enzymes, which degrade PAT to form ascladiol. The degradation products of PAT by XZ1 were identified as ascladiol and desoxypatulinic acid, which was then complete degraded to form unknown final degradation products. Toxic analyses on Caco-2 cells showed that the ascladiol, desoxypatulinic acid and the final degradation products were significantly less toxic compared to PAT, which was mainly manifested in less influence on cell vitality, cell integrity and reactive oxygen species accumulation compared to PAT. Finally, the results revealed the PAT degradation enzyme, as well as the safety of the degradation products, which provide basis for the future application of this yeast to decontamination of PAT in food.