Diphtheria toxin (DT) is one of the most extensively studied bacterial toxins with intracellular action. It is produced by toxigenic strains of Corynebacterium diphtheriae and is responsible for the symptoms of diphtheria. The toxin was isolated in 1888. A vaccine was developed in 1923. The crystal structure of the toxin was solved in 1992, bringing tremendous progress in understanding the molecular events involved in toxicity. DT contains three structural domains, each carrying a distinct biological function implicated in the intoxication of the cell: cell-surface binding and internalization into endosomes, crossing of the endosome membrane into the cytosol and blocking of cellular protein synthesis. All three domains of the DT, isolated or combined with other proteins, are now exploited for their biological properties to design biotechnological tools and therapeutics.