Cell-penetrating peptides (CPPs) are promising tools for the transfection of various substances, including nucleic acids, into cells. The aim of the current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed from CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed the effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed by the addition of the ions, without increasing cytotoxicity. We propose a novel strategy for preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, with the aim of increasing CPP-mediated transfection efficiency and the likelihood of using CPPs in clinics.