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The present work investigates in vitro the delivery of the anticancer drug mitoxantrone (MTX) to HeLa cancer cells by means of polyethylene glycol (PEG) liposomes functionalized with the novel cell penetrating peptide gH625. This hydrophobic peptide enhances the delivery of doxorubicin (Doxo) to the cytoplasm of cancer cells, while the mechanism of this enhancement has not yet been understood. Here, in order to get a better insight into the role of gH625 on the mechanism of liposome-mediated drug delivery, we treated HeLa cells with liposomes functionalized with gH625 and loaded with MTX; functionalized and not liposome were characterized in terms of their physico-chemical properties and drug release kinetics. To quantify the MTX uptake and to study the subcellular drug distribution and interaction, we took advantage of the intrinsic fluorescence of MTX and of the fluorescence-based techniques like fluorescence-activated cell sorting (FACS) and confocal spectral imaging (CSI). FACS data confirmed that gH625 increases the total intracellular MTX content. CSI data indicated that when liposomes are decorated with gH625 an enhanced staining of the internalized drug is observed mainly in hydrophobic regions of the cytoplasm, where the increased presence of an oxidative metabolite of the drug is observed. The cytotoxicity on HeLa cell line was higher for functionalized liposomes within 4–6 h of treatment. To summarise, the MTX delivery with gH625-decorated nanoliposomes enhances the quantity of both the intracellular drug and of its oxidative metabolite and contributes to higher anticancer efficacy of the drug at the delay of 4–6 h.