In burn patients, wound healing is often accompanied by hypertrophic scarring (HTS), resulting in both functional and aesthetic problems. HTSs are characterized by abundant presence of myofibroblasts (MFs) residing in the dermis. HTS development and MF persistence is primarily regulated by TGF-β signalling. A promising method to target the transforming growth factor receptor I (TGFβRI; also known as activin-like kinase 5 (ALK5)) is by making use of exon skipping through antisense oligonucleotides. In HTS the distinguishing border between the papillary dermis and the reticular dermis is completely abrogated, thus exhibiting a one layered dermis containing a heterogenous fibroblast population, consisting of papillary fibroblasts (PFs), reticular fibroblasts (RFs) and MFs. It has been proposed that PFs, as opposed to RFs, exhibit anti-fibrotic properties. Currently, it is still unclear which fibroblast subtype is most affected by exon skipping treatment. Therefore, the aim of this study was to investigate the effect of TGFβRI inhibition by exon skipping in PF, RF and HTS fibroblast monocultures. Morphological analyses revealed the presence of a PF-like population after exon skipping in the different fibroblast cultures. This observation was further confirmed by the expression of genes specific for PFs, demonstrated by qPCR analyses. Further investigations on mRNA and protein level revealed that indeed MFs and to a lesser extent RFs are targeted by exon skipping. Furthermore, collagen gel contraction analysis showed that ALK5 exon skipping reduced TGF-β- induced contraction together with decreased alpha-smooth muscle actin expression levels. In conclusion, we show for the first time that exon skipping primarily targets pro-fibrotic fibroblasts. This could be a promising step towards reduced HTS development of burn tissue.