As aortic valve stenosis (AVS) progresses, the valve tissue also stiffens. This increase in tissue stiffness causes the valvular interstitial cells (VICs) to transform into myofibroblasts in response. VIC-to-myofibroblast differentiation is critically involved in the development of AVS. Herein, we investigated the role of mechanosensitive Ca-permeant transient receptor potential vanilloid 4 (Trpv4) channels in matrix stiffness- and transforming growth factor β1 (TGFβ1)-induced VIC-myofibroblast activation. We confirmed Trpv4 functionality in primary mouse wild-type VICs compared to Trpv4 null VICs using live Ca influx detection during application of its selective agonist and antagonist. Using physiologically relevant hydrogels of varying stiffness that respectively mimic healthy or diseased aortic valve tissue stiffness, we found that genetic ablation of Trpv4 blocked matrix stiffness- and TGFβ1-induced VIC-myofibroblast activation as determined by changes in morphology, alterations of expression of α-smooth muscle actin, and modulations of F-actin generation. Our results showed that N-terminal residues 30-130 in Trpv4 were crucial for cellular force generation and VIC-myofibroblast activation, while deletion of residues 1-30 had no noticeable negative effect on these processes. Collectively, these data suggest a differential regulatory role for Trpv4 in stiffness/TGFβ1-induced VIC-myofibroblast activation. Our data further showed that Trpv4 regulates stiffness/TGFβ1-induced PI3K-AKT activity that is required for VIC-myofibroblast differentiation and cellular force generation, suggesting a mechanism by which Trpv4 activity regulates VIC-myofibroblast activation. Altogether, these data identify a novel role for Trpv4 mechanotransduction in regulating VIC-myofibroblast activation, implicating Trpv4 as a potential therapeutic target to slow and/or reverse AVS development.