Cardiac fibrosis remains a significant and currently untreatable contributor to mortality in chronic heart disease. This study aimed to comprehensively map cell-specific pathological changes in fibrotic heart tissue from aortic stenosis patients using single-nucleus RNA sequencing (snRNA-seq). We specifically sought to identify novel cell-specific candidate genes involved in cardiac fibrosis and validate their functional impact using both in vitro and in vivo models. Our ultimate goal was to enhance the understanding of fibrogenesis in aortic stenosis, thereby guiding the development of targeted antifibrotic therapies. We conducted single nucleus RNA sequencing (snRNA-seq) on human fibrotic aortic stenosis and non-failing donor heart samples. The final dataset consisted of 41 330 nuclei distributed across 28 cell clusters representing 6 major cell types. SnRNA-seq revealed a disease-specific subpopulation of fibroblasts, characterized by the presence of both anti-fibrotic and pro-fibrotic markers. Interestingly, TCF21, a basic helix-loop-helix transcription factor, was identified as a potential key regulator within this distinct fibroblast population, and we performed functional studies to identify its role in disease progression accordingly. Loss of TCF21 in cardiac fibroblasts caused increased myofibroblast-like gene expression and differentiation, whereas upregulation of TCF21 in cardiac fibroblasts exerted an anti-fibrotic effect. Adeno-associated virus-mediated cardiac fibroblast-specific knockdown and overexpression in vivo further demonstrate that TCF21 improves cardiac remodelling. This investigation highlights the role of TCF21 as a protective regulator in cardiac fibrosis. Moreover, the upregulation of TCF21 holds promise as a strategy for treating cardiac fibrosis.