10.1002/anie.201705489 Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes. These include i) PsiD which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, that catalyzes the phosphotransfer step, iii) the methyl transferase PsiM, catalyzing iterative N-methyltransfer as terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production.