<jats:title>Summary</jats:title><jats:sec><jats:label/><jats:p>The important anticancer drugs, vinblastine, vincristine and analogs, are composed of the monoterpenoid indole alkaloids (<jats:styled-content style="fixed-case">MIA</jats:styled-content>s), catharanthine and vindoline, found uniquely in the medicinal plant, <jats:italic>Catharanthus roseus</jats:italic>. While 26 genes involved in the assembly of these two <jats:styled-content style="fixed-case">MIA</jats:styled-content>s are known, two key reactions have eluded characterization to complete the documentation of the vinblastine pathway in this plant species. The assembly of these dimeric <jats:styled-content style="fixed-case">MIA</jats:styled-content>s requires O‐acetylstemmadenine oxidase (<jats:styled-content style="fixed-case">ASO</jats:styled-content>) and a dual function geissoschizine synthase (<jats:styled-content style="fixed-case">GS</jats:styled-content>) that reduces cathenamine to form geissoschizine, and that also reduces the <jats:styled-content style="fixed-case">ASO</jats:styled-content> product to form a common intermediate for subsequent conversion by four separate hydrolases to catharanthine, tabersonine or vincadifformine, respectively. The <jats:italic>in planta</jats:italic> role of <jats:styled-content style="fixed-case">ASO</jats:styled-content> is supported by identifying a single amino acid‐substituted <jats:styled-content style="fixed-case">ASO</jats:styled-content> mutant with very low enzyme activity and by virus‐induced gene silencing of <jats:styled-content style="fixed-case">ASO</jats:styled-content> to produce plants that accumulate O‐acetylstemmadenine rather than catharanthine and vindoline found in wild‐type (<jats:styled-content style="fixed-case">WT</jats:styled-content>) plants. The <jats:italic>in planta</jats:italic> role of <jats:styled-content style="fixed-case">GS</jats:styled-content> is supported by showing that a low <jats:styled-content style="fixed-case">GS</jats:styled-content>‐expressing mutant accumulating lower levels of catharanthine and vindoline also displays significantly lower tabersonine‐forming activity in coupled enzyme assays than in the <jats:styled-content style="fixed-case">WT</jats:styled-content> background. Gene expression analyses demonstrate that both <jats:styled-content style="fixed-case">ASO</jats:styled-content> and <jats:styled-content style="fixed-case">GS</jats:styled-content> are highly enriched in the leaf epidermis where the pathways for catharanthine and tabersonine biosynthesis are expressed. The full elucidation of this canonical pathway enables synthetic biology approaches for manufacturing a broad range of <jats:styled-content style="fixed-case">MIA</jats:styled-content>s, including these dimers used in cancer treatment.</jats:sec>