Abstract: Camptothecin is one of the most commonly used anticancer drugs worldwide, yet the downstream biosynthetic route from strictosidine to camptothecin has remained unclear for more than half a century. Here, we searched for proteins involved in camptothecin biosynthesis from the camptothecin-producing plant Ophiorrhiza pumila by chemoproteomics and identified OpCYP716E111. Exogenously expressing OpCYP716E111 in Nicotiana benthamiana and yeast (Saccharomyces cerevisiae) led to the production of strictosamide epoxide 2 from strictosamide 1. Our findings thus reveal the enzyme responsible for the biosynthesis of strictosamide epoxide in the camptothecin biosynthetic pathway. In addition, our results highlight the potential of using chemoproteomics as a tool for discovering enzymes involved in natural product biosynthesis.