Abstract Body (Do not enter title and authors here): Cardiac fibrosis arises from the abnormal activation of cardiac fibroblasts (CFs) in response to both chemical and mechanical stressors. While extracellular matrix (ECM) stiffness is a key determinant of fibroblast behavior, the molecular mechanisms linking mechanical signals to gene expression remain poorly understood. To address this gap, we developed a three-dimensional (3D) hydrogel system that mimics the ECM stiffness of normal, mid-stage, and fibrotic myocardium. Using RNA sequencing, we identified mechanosensitive genes in CFs cultured within this system. Weighted gene co-expression network analysis (WGCNA) revealed a 98-gene cluster, encompassing PCSK6, ATP8B4, FILIP1L, and ARF2 among others, which was significantly upregulated across stiffness gradients. Single-cell RNA sequencing from myocardial infarction and pressure overload-induced cardiac fibrosis models validated the mechanosensitivity of these genes, uncovering distinct temporal expression patterns under acute versus chronic mechanical stress. Notably, the marked upregulation of this gene cluster in human dilated and hypertrophic cardiomyopathy samples underscores its clinical relevance. Collectively, our findings deepen the understanding of the mechanobiology underlying cardiac fibrosis and highlight potential diagnostic markers and therapeutic targets for modulating mechanical stress in this pathological condition.