Cardiac myocytes are continuously exposed to shear stress during cardiac cycle. We have previously reported that shear stress enhances Ca²⁺ transients in rat ventricular myocytes. Here we examined the underlying mechanism for the shear-induced enhancement in Ca²⁺ signaling in isolated murine ventricular myocytes using confocal Ca²⁺ imaging combined with fluid micro-jet system. Application of shear stress of about 16 dyn/cm² to single rat ventricle cell enhanced Ca²⁺ transient magnitude by ~35% at steady state (15 s), with an early transient increase in diastolic Ca²⁺ level. Inhibitions of pannexin (Panx) channels and external ATP action with probenecid and apyrase, respectively, significantly inhibited the stimulatory effects by shear stress. The P2Y1 purinoceptor antagonist (MRS2179) or IP₃ receptor blocker (2-APB) significantly suppressed shear-mediated prolonged enhancement in Ca^2+​ transients. In ventricular myocytes from P2Y1 purinoceptor knockout mice, shear stress-induced prolonged stimulatory effects on Ca²⁺ transients were largely reduced, with an increase of early-phase basal Ca²⁺ signal. Our data suggest that shear stress augments depolarization-induced Ca²⁺ release via P2Y1 receptor-IP₃ receptor signaling pathway, activated by pannexin-mediated ATP release in autocrine mode.