Manipulation of disease susceptibility (S) genes represent a credible strategy to achieve more durable disease resistance in crops. Daunting challenges remain in cloning hidden wheat S genes for stripe rust, a leading food crop disease caused by Puccinia striiformis f. sp. tritici (Pst). Here, we unrevealed that a wheat zeaxanthin epoxidase (wZEP) as an ideal precise regulation target to boost Pst-resistance. We isolated the wheat increased stripe rust endurance 1 (wise1) mutant in which a premature stop mutation in wZEP-2B underpinned the enhanced Pst-resistance phenotype. Genetic analyses revealed a fueled ROS accumulation in wzep mutants and demonstrated the correlation between wZEP dysfunction and enhanced Pst-resistance. Moreover, wZEP participated in the adult-plant Pst-resistance of Wheat Kinase START 1 (WKS1, YR36), which bound, phosphorylated, and diminished wZEP’s biochemical activity. A rare natural missense variation in wZEP attenuated its activity and Pst-resistance in a natural wheat population with negotiable adverse effect on wheat agricultural traits. Our study identified wZEP as a hidden S gene for Pst-resistance in wheat and extended our knowledge about the zeaxanthin’s regulation and biological function in crop disease resistance. We predicted that wZEP worked together with PsbO and tAPX to promote the ROS burst during Pst-resistance. This work opened the door to stack the valuable wZEP genetic materials with other known Pst-resistance genes to alleviate food spoilage to nourish the world’s population.