Abstract Body: Dimorphic ion channels, chloride intracellular ion channels (CLIC), are known to regulate several cellular processes, but their implication in cardiac physiology remains unclear. Artificial planar lipid bilayer studies have shown that CLIC2 negatively modulates the activity of cardiac ryanodine receptor 2 (RyR2), implicating the sarcoplasmic/endoplasmic reticulum (SR/ER) Ca²⁺ release. In heart failure, RyR2 channel activity is altered causing aberrant SR Ca²⁺ release, depletion of SR Ca²⁺ stores, and reduced myocardial contractility in heart failure. Hence, the characterization of intrinsic RyR2 modulators will represent a novel target for the prevention and treatment of heart failure. Moreover, CLIC2 protein expression is significantly increased (p<0.05) in end-stage failing human heart samples compared to non-failing heart tissues. Hence using human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM), we assessed the contribution of CLIC2 in the regulation of cellular Ca²⁺ homeostasis in cardiac physiology. CLIC2 knockout hiPSC-CM (CLIC2KO-CMs) showed altered electrophysiological properties, where the beat period (0.686±0.018 sec) and field potential duration (134.266±9.07 ms) decreased as compared to age-matched WT hiPSC-CM (WT-CMs) (0.760±0.014sec and 215.37±7.82ms, respectively). Similarly, spike amplitude showed a decrease in CLIC2KO-CMs (0.339 ± 0.046 mV) compared to WT-CMs (0.705±0.069 mV). Moreover, the APD30, APD50, and APD90 in CLIC2KO-CMs (0.109±0.013, 0.132±0.011, 0.186±0.008 sec respectively) were significantly reduced as compared to the WT-CMs (0.173±0.005, 0.204±0.008, 0.264±0.008 sec respectively). Furthermore, the intracellular Ca²⁺ measurements revealed an altered Ca²⁺ handling in CLIC2KO-CMs, where a decrease in peak amplitude (3.35±0.13) and duration (1179±9.52 ms) was observed compared to the WT-CMs (5.87±0.25 and 3081.38±55.95 ms, respectively). Finally, the caffeine-sensitive Ca²⁺ store load in CLIC2KO-CMs showed a marked reduction in cytosolic caffeine transient amplitude, suggesting an increased SR-Ca²⁺ leak due to hyperactive RyR2 channels. Overall, our study highlights CLIC2 as an understudied negative modulator of RyR2 involved in the SR/ER Ca²⁺ release mechanism that is upregulated in failing human hearts.