Background: Cancer associated fibroblasts (CAFs), are known to promote stromal-epithelial paracrine interactions, stromal remodelling and angiogenesis, within the tumour microenvironment. In oral squamous cell carcinomas, elevated expression of a myofibroblast CAF marker, alpha-smooth muscle actin (αSMA) and the matricellular proteoglycan versican are both predictive of invasive phenotypes and poor prognosis. The molecular mechanisms underlying myofibroblast transdifferentiation are poorly characterised. Here, the role of microRNA-145, a small non-coding RNA which negatively regulates multiple gene transcripts, was investigated in stromal fibroblasts. Methods: Normal oral fibroblasts (NOFs) and fibroblasts isolated from OSCC (CAFs) were treated with cancer cell line conditioned medium and TGF-β1, and the expression of miR-145, versican and myofibroblasts markers αSMA, collagen-1a (COL1A1), and fibronectin-1 with extra domain A (FN1-EDA) were assessed by qRT-PCR, immunoblotting and immunocytochemistry. A synthetic precursor miR-145 was used to overexpress miR-145 in fibroblasts prior, or subsequent to, TGF-β1 treatment and the myofibroblast phenotype was investigated by assessing molecular markers, cell contractility and the ability to promote paracrine cancer cell migration. Versican expression was investigated and loss of function experiments were used to investigate its effect on myofibroblast phenotype. Putative genes involved in myofibroblast regulation were also assessed by qRT-PCR. Similar experiments were performed in primary dermal fibroblasts. Results: CAFs had a significantly higher miR-145 expression than NOFs, but no difference in myofibroblast markers was observed. TGF-β1 induced the expression of miR-145 and myofibroblast markers, and promoted contractility and cancer cell paracrine migration. miR-145 gain of function experiments attenuated and rescued the TGF-β1-induced myofibroblast phenotype. miR-145 negatively regulated versican, and versican loss of function had a small effect on myofibroblast phenotype. miR-145 and versican had similar effects in dermal fibroblasts. Conclusions: miR-145 inhibits and reverses oral and dermal myofibroblast transdifferentiation. Therefore, exogenously delivering miR-145 to the tumour microenvironment and fibrotic areas could potentially treat deleterious myofibroblasts.