Matrix elasticity regulates proliferation, apoptosis, and differentiation of many cell types across various tissues. In particular, stiffened matrix in fibrotic lesions has been shown to promote pathogenic myofibroblast activation. To better understand the underlying pathways by which fibroblasts mechano-sense matrix elasticity, we cultured primary valvular interstitial cells (VICs) isolated from porcine aortic valves on poly(ethylene glycol)-based hydrogels with physiologically relevant and tunable elasticities. We show that soft hydrogels preserve the quiescent fibroblast phenotype of VICs much better than stiff plastic plates. We demonstrate that the PI3K/AKT pathway is significantly upregulated when VICs are cultured on stiff gels or tissue culture polystyrene compared with freshly isolated VICs. In contrast, myofibroblasts de-activate and pAKT/AKT decreases as early as 2 h after reducing the substrate modulus. When PI3K or AKT is inhibited on stiff substrates, myofibroblast activation is blocked. When constitutively active PI3K is overexpressed, the myofibroblast phenotype is promoted even on soft substrates. These data suggest that valvular fibroblasts are sensing the changes in matrix elasticity through the PI3K/AKT pathway. This mechanism may be used by other mechano-sensitive cells in response to substrate modulus, and this pathway may be a worthwhile target for treating matrix stiffness-associated diseases. Furthermore, hydrogels can be designed to recapitulate important mechanical cues in native tissues to preserve aspects of the native phenotype of primary cells for understanding basic cellular responses to biophysical and biochemical signals, and for tissue-engineering applications.mechanosensing | tissue stiffening | phosphatidylinositide 3-kinase signaling E very cell has its distinct microenvironment. Fibroblasts reside in the interstitial mesenchyme, maintaining the balance and structure of the matrix; muscle satellite cells reside between the basal lamina and muscle fibers, waiting to be activated upon injury. In a basic sense, the cellular microenvironment is defined as the extracellular space surrounding and supporting the cell. This microenvironment is often comprised of extracellular matrix (ECM), soluble chemical factors, and neighboring cells. The ECM in particular does not just act as a passive scaffold, but instructs cell fate through the coordinated and dynamic presentation of biochemical and biophysical cues. For example, in fibrotic tissues stiffened by excessive collagen deposition, resident fibroblasts acquire an activated myofibroblast phenotype with α-smooth muscle actin (αSMA) stress fibers (1). The integrity and signaling of the ECM are essential for regulating normal cellular function and maintaining organ homeostasis.The aortic valve, which controls the unidirectional flow of blood during heart contractions, is composed of elastin-, proteoglycanand collagen-rich layers of ECM (2) and supports survival and metabolism of valvular interstitial cells (VICs). VICs are the main fib...