Diphtheria toxin remains one of the most successfully studied of the bacterial protein toxins. A detailed understanding of the structure function relationships of the toxin and the role of each domain in the intoxication process is well understood. This understanding has led to the development of diphtheria toxin-related fusion protein toxins which are targeted toward specific cell surface receptors. The first of these targeted toxins are now approved for human clinical use in the treatment of refractory hematologic malignancies and graft-versus-host disease. In recent years, studies on the molecular mechanism by which the diphtheria toxin catalytic domain is translocated across the early endosomal membrane has revealed that a host cell cytosolic translocation factor complex facilitates the entry process. A detailed understanding of this process will further stimulate the development of new approaches toward the delivery of cargo, ranging from protein to nucleic acid and/or protein nucleic acids, to the eukaryotic cell cytosol.