<p>Low molecular weight protamines (LMWPs) are polycationic peptide fragments that possess a high capability of penetrating cells with low toxicity. These peptides have been utilized to produce innovative pharmaceutical formulations for gene delivery systems and nanopharmaceutical particles. Despite the potential impact of LMWPs for therapeutic applications their subcellular trafficking pathways, particularly the translocation to the nuclear/nucleolar region, is not completely understood. Targeting delivery of drugs to these compartments could be utilized to treat diseases like breast cancer to increase drug efficiency, reduce side toxicity and overcome drug resistance mechanisms. In this study, the dynamic trafficking of the LMWP fragment (Pep2) to the nuclear/nucleolar compartment has been analyzed in breast cancer cell lines using laser scanning confocal microscopy and live-cell imaging techniques. Also, the present study determines a key part of the LMWP trafficking: a potential nuclear/nucleolar localization sequence (NLS/NoLS) that could be associated with the peptide. Indeed, we have identified the specific sequence (RRRRRGGRRRR) as a potential NLS/NoLS that could be required by the LMWP fragment (Pep2) for its trafficking and accumulation in the nuclear/nucleolar compartments. The functionality of the sequence has been validated using mutagenesis experiments combined with microscopic analysis in cells. The findings of this study could be utilized to improve the efficiency of various LMWP-containing biopharmaceutical formulations, probably through modulation of their subcellular localization.</p>