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Macromolecular biomolecules are currently dethroning classical small molecule therapeutics because of their improved targeting and delivery properties. Protamine-a small polycationic peptide-represents a promising candidate. In nature, it binds and protects DNA against degradation during spermatogenesis due to electrostatic interactions between the negatively charged DNA-phosphate backbone and the positively charged protamine. Researchers are mimicking this technique to develop innovative nanopharmaceutical drug delivery systems, incorporating protamine as a carrier for biologically active components such as DNA or RNA. The first part of this review highlights ongoing investigations in the field of protamine-associated nanotechnology, discussing the self-assembling manufacturing process and nanoparticle engineering. Immune-modulating properties of protamine are those that lead to the second key part, which is protamine in novel vaccine technologies. Protamine-based RNA delivery systems in vaccines (some belong to the new class of mRNA-vaccines) against infectious disease and their use in cancer treatment are reviewed, and we provide an update on the current state of latest developments with protamine as pharmaceutical excipient for vaccines.