The potent transcription inhibitor Actinomycin D is used with several cancers. Here, we report the discovery that this naturally occurring antibiotic inhibits two human neutral aminopeptidases, the cell-surface alanine aminopeptidase and intracellular methionine aminopeptidase type 2. These metallo-containing exopeptidases participate in tumor cell expansion and motility and are targets for anticancer therapies. We show that the peptide portions of Actinomycin D and Actinomycin X are not required for effective inhibition, but the loss of these regions changes the mechanism of interaction. Two structurally less complex Actinomycin D analogs containing the phenoxazone chromophores, Questiomycin A and Actinocin, appear to be competitive inhibitors of both aminopeptidases, with potencies similar to the non-competitive macrocyclic parent compound ( in the micromolar range). The mode of action for all four compounds and both enzymes was demonstrated by molecular modeling and docking in the corresponding active sites. This knowledge gives new perspectives to Actinomycin D's action on tumors and suggests new avenues and molecules for medical applications.