Pancreatic cancer remains a highly malignant disease with limited effective treatment options. Covalent-binding drugs have received substantial attention due to their high specificity, selectivity, and low resistance potential. In this study, we demonstrated that jolkinolide B (JB), a natural epoxide compound, exerts potent anti-tumor effects against pancreatic cancer both in vitro and in vivo. By utilizing high-resolution protein identification and quantitative technology of mass spectrometry, we revealed the logical relationship between JB, target, mechanism, and phenotype, which makes JB a promising lead compound for further drug development.