Abstract Dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiphenyl-dichloroethylene (DDE) are ubiquitously found in the environment and linked to cardiovascular diseases—with a majority of the work focused on hypertension. Studies investigating whether DDx can interact with molecular targets on cardiac tissue to directly affect cardiac function are lacking. Therefore, we investigated whether o,pʹ-DDT, p,pʹ-DDT, o,pʹ-DDE, or p,pʹ-DDE (DDx, collectively) can directly alter the function of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) by assessing their effect(s) on hiPSC-CMs Ca2+ dynamics. DDx (0.1–10 µM) affected hiPSC-CMs synchronous Ca2+ oscillation frequency in a concentration-dependent manner, with p,pʹ-DDT and p,pʹ-DDE also decreasing Ca2+ stores. HEK-RyR2 cells cultured under antibiotic selection to induce expression of wild-type mouse ryanodine receptor type 2 (RyR2) are used to further investigate whether DDx alters hiPSC-CMs Ca2+ dynamics through engagement with RyR2, a protein critical for cardiac muscle excitation-contraction coupling (ECC). Acute treatment with 10 µM DDx failed to induce Ca2+ release in HEK293-RyR2, whereas pretreatment with DDx (0.1–10 µM) for 12- or 24-h significantly decreased sarcoplasmic reticulum Ca2+ stores in HEK-RyR2 cells challenged with caffeine (1 mM), an RyR agonist. 3Hryanodine-binding analysis using murine cardiac RyR2 homogenates further confirmed that all DDx isomers (10 µM) can directly engage with RyR2 to favor an open (leaky) confirmation, whereas only the DDT isomers (10 µM) modestly (≤10%) inhibited SERCA2a activity. The data demonstrate that DDx increases heart rate and depletes Ca2+ stores in human cardiomyocytes through a mechanism that impairs RyR2 function and Ca2+ dynamics. Impact Statement DDT/DDE interactions with RyR2 alter cardiomyocyte Ca2+ dynamics that may contribute to adverse cardiovascular outcomes associated with exposures.