The broad use of oligonucleotides (ON) in therapeutic and biotechnological applications is limited due to inefficient delivery methods. In parallel with lipids and polymeric carriers, cell-penetrating peptides (CPPs) are efficient vehicles for delivering nucleic acids of various types and activity into cells. In the current work, we examined the structural motifs required for the high efficacy of PepFect14, an often-used CPP for ON delivery, by introducing point mutations into the peptide sequence. We predicted the characteristics of modified CPPs, and analyzed their structure and ability to condense ONs into nanoparticles (NPs) using biophysical methods. We evaluated the ability of new PF14 analogs to deliver splicing switching oligonucleotides (SCO) and small interfering RNA (siRNA) in reporter cell lines, as well as microRNA miR-146a in human primary keratinocytes and in a mouse skin inflammation in vivo. Our findings indicate that the α-helical structure of PF14 is essential for efficient ON delivery, and mutations that disrupt the hydrophobic or cationic face in the peptide abolish NP formation and cellular internalization. PF14-Lys, an analog containing lysine residues instead of original ornithines, yielded a higher biological response to SCO and siRNA in the respective reporter cells than PF14. Furthermore, PF14-Lys efficiently delivered miRNA into keratinocytes and led to the subsequent downregulation of the target genes. Importantly, subcutaneously administered PF14-Lys-miR-146a NPs suppressed the inflammatory responses in mouse model of irritant contact dermatitis. In conclusion, our results suggest that PF14-Lys is a highly promising delivery vector for various oligonucleotides, applicable both in vitro and in vivo.