Background <p>Mitral regurgitation is highly prevalent and elevates the risk of heart failure. Regurgitant flow induced shear stress disrupts Ca²⁺ homeostasis in atrial cardiomyocytes. Overloaded Ca²⁺ is a crucial regulator of endoplasmic reticulum stress-mediated apoptosis, although the regulatory mechanisms remain unclear. We aimed to explore the relationship between shear stress and Ca²⁺ homeostasis.</p> Methods and results <p>Through employing an in vitro model replicating atrial cardiomyocytes during mitral regurgitation, we observed that shear stress increased Ca²⁺ oscillation and the amplitude of Ca²⁺ waves through extracellular Ca²⁺ influx. Suppression of mechanosensitive Ca²⁺ channels or store-operated calcium entry resulted in decreased intracellular Ca²⁺ concentration and oscillation. Conversely, the inhibition of voltage-gated Ca²⁺ channels did not significantly impact the Ca²⁺ concentration. Remarkably, shear stress elevated the expression of endoplasmic reticulum stress and apoptosis-related proteins in HL-1 cells in a time-dependent manner. Also, in a novel rodent mitral regurgitation model, shear stress increased apoptosis and endoplasmic reticulum stress expression compared to sham rats.</p> Conclusions <p>Our findings underscore that shear stress induces apoptosis through mechanosensitive and store-operated Ca²⁺ channels, leading to Ca²⁺ overload and endoplasmic reticulum stress. Understanding the mechanisms behind shear stress-disturbed Ca²⁺ homeostasis may enhance the treatment of mitral regurgitation-induced heart failure.</p>