Significance Euphorbia peplus , a member of the Euphorbia genus, is rich in jatrophane and ingenane diterpenoids. Using a metabolomics-guided transcriptomic approach to gene candidate identification, we have discovered a short-chain dehydrogenase gene involved in the production of the lathyrane jolkinol E. We have developed a virus-induced gene-silencing method in E. peplus that has allowed us to demonstrate the direct relationship between casbene and polycyclic diterpenoids and that jolkinol C acts as a key branch point intermediate in the production of ingenanes and jatrophanes. This work contributes both knowledge and tools for engineering production of bioactive diterpenoids in heterologous host systems, thus enabling their further evaluation and development.