Reprogramming of cancer cell metabolism underpins several important tumourigenic processes, including proliferation, metastasis and therapeutic resistance. In addition, there is growing evidence that non-cancer cell types within the tumour microenvironment may also undergo metabolic reprogramming so as to support cellular processes that facilitate tumour growth. Cancer-associated fibroblasts (CAFs) are a class of non-cancer cell that are activated in response to diverse cues within the tumour niche and play numerous roles in tumour progression. Notably, a subclass of TGFβ-induced CAFs known as myoCAFs play a critical role in depositing and remodelling collagenous extracellular matrix (ECM) in the tumour microenvironment. This myoCAF-mediated dysregulation of ECM architecture is a key feature of most solid tumours, and promotes pro-oncogenic signalling and local invasion of cancer cells. Although ECM production is known to be a metabolically demanding process, the specific metabolic pathways that are reprogrammed during myoCAF activation are poorly understood. Using an in vitro model of mammary myoCAF activation, we have interrogated metabolic and transcriptomic changes that occur during the process of TGFβ-mediated CAF induction. Using RNAseq, immunofluorescence microscopy and biochemical analyses, we found that cholesterol biosynthesis is upregulated in CAFs and that CAFs exhibit specific enrichment for cholesterol in the endoplasmic reticulum (ER) membrane. In addition, ER mass is increased in CAFs relative to normal fibroblasts, thereby facilitating their ECM-secreting and remodelling function. Intriguingly, we find that pharmacological inhibition of cholesterol biosynthesis diminishes CAF ER mass and suppresses CAF ECM remodelling capacity. Together, these data suggest that reprogramming of cholesterol facilitates CAF ER function and underlies the activation of normal mammary fibroblasts to myoCAFs. By better understanding this mechanism of CAF activation, we hope to elucidate strategies for targeting tumour progression at the microenvironmental level.