Lung squamous cell carcinoma (SCC), accounting for approximately 30% of non–small cell lung cancer, is often refractory to therapy. Screening a small-molecule library, we identified digitoxin as a high potency compound for suppressing human lung SCC growth in vitro and in vivo. Mechanistic investigations revealed that digitoxin attenuated YAP phosphorylation and promoted YAP nuclear sequestration. YAP activation led to excessive accumulation of reactive oxygen species (ROS) by downregulating the antioxidant enzyme GPX2 in a manner related to p63 blockade. In patient-derived xenograft models, digitoxin treatment efficiently inhibited lung SCC progression in correlation with reduced expression of YAP. Collectively, our results highlight a novel tumor-suppressor function of YAP via downregulation of GPX2 and ROS accumulation, with potential implications to improve precision medicine of human lung SCC.