Taxol (paclitaxel) is a widely used anti-cancer drug with a complex biosynthetic pathway that has puzzled biochemists for decades. Owing to inefficient chemical synthesis, Taxol supply depends on costly semi-synthesis. Elucidating the Taxol biosynthesis will solve a long-standing question in biochemistry and enable cost-effective production using biotechnological methods. While recent advances have improved our understanding of the steps leading up to the intermediate baccatin III, the final steps of the pathway remain elusive. Here we use gene co-expression analysis, chemically synthesized intermediates and a stepwise learning-by-building approach to reveal the enzymes that catalyse the final two modifications, that is, C2′α hydroxylation and 3′-N benzoylation, which are essential for Taxol’s bioactivity. To replace the current semi-synthetic method of Taxol production, we reconstruct the late pathway in yeast and synthesize Taxol from the readily available intermediate baccatin III. This work provides a complete understanding of Taxol biosynthesis and establishes a foundation for its biotechnological production.