Fibrosis is the result of an imbalance in the deposition and degradation of extracellular matrix (ECM), with the result being deposition of excessive and dysfunctional ECM components. Fibrosis can occur as part of the wound repair process to form scar tissue but also as a progressive fibrotic disease. Fibrosis therefore presents a significant clinical problem. One of the most important proteins influencing ECM architecture in the fibrotic scar is collagen. Regulating the deposition of excess collagen either during the acute or prolonged remodeling phase after injury, or as fibrosis progresses in disease, has the potential to alter collagen architecture and improve tissue appearance and function. It is important to characterize the regulatory elements and transcription factors that regulate type one-collagen gene expression to be able to modulate the collagen deposition and hence regulate fibrosis. G-quadruplexes (G4) are non-canonical DNA secondary structures that can form from certain guanine–rich sequences. They are over-represented in regions associated with regulation in the human genome (e.g. gene promoters, enhancers and 3’-end of telomeric DNA) and their formation has been implicated in transcriptional regulation, recombination events and genomic instability. G4s are stabilized by the presence of cations such as Calcium (Ca²⁺), and it is well know that fibrosis is associated with a loss of Ca2+ homeostasis. The aim of this study is to investigate the occurrence of G4-DNA in a fibroblast scar model to determine the relationship between alterations in G4 formation and scarring. To do so, we established a fibrotic scar model using primary human fibroblasts derived from a dupuytren patient (progressive fibrotic disorder), and stimulated these cells with TGF-β (in-vitro scar model). Fibroblasts stimulated with TGF-β displayed significant increases (p< 0.05) in: collagen immunoreactivity and collagen I mRNA expression verifying scar confirmation; intracellular calcium ion concentration and increased G4-DNA formation, detected by immunohistochemical analysis with a G4-specific antibody (BG4). Furthermore, G4-motifs were identified in the Col 1 gene, and these motifs were analysed by biophysical studies to confirm G4-DNA formation. Our findings suggest a relationship between alterations in G4-DNA formation and scarring which is associated with a loss in calcium homeostasis. This work is a first step towards targeting G-quadruplexes as a therapeutic strategy for external control of gene expression in order to regulate ECM deposition and create a permissible environment for repair within the scar.