Antisense oligonucleotides (ASOs) that bind target pre-mRNA with high affinity have been shown to alter splicing patterns and offer promise as therapeutics. Previous studies have shown that ASOs fully modified with 2'-O-methoxyethyl (2'-O-MOE) sugar residues redirect constitutive and alternative splicing of the murine interleukin-5 receptor-alpha (IL-5Ralpha) chain pre-mRNA in cells, resulting in inhibition of the membrane-bound isoform and enhanced expression of the soluble isoform. Here, we show that antisense peptide nucleic acids (PNAs) alter splicing of the IL-5Ralpha pre-mRNA in a fashion similar to their 2'-O-MOE-modified counterparts of the same sequence. Moreover, using PNA as the splicing modulator, the length of the antisense oligomer could be shortened from 20 to 15 nucleobase units to obtain a comparable effect. Treatment of cells with antisense PNA resulted in dose-dependent, specific downregulation of IL-5Ralpha membrane isoform mRNA expression and enhanced levels of the soluble IL-5Ralpha isoform transcript, with an EC50 equivalent to that observed in parallel with the corresponding 2'-O-MOE ASO. The pronounced activity of antisense PNAs in modulating IL-5Ralpha mRNA splicing observed in our study identifies these compounds as a promising new class of lower molecular weight splicing modulators.