Mutations that cause truncation of cardiac myosin binding protein C (cMyBP-C) are common causes of hypertrophic cardiomyopathy (HCM), and cause disease through a mechanism of haploinsufficiency. Recent studies in isogenic iPSC-cardiomyocyte (CM) heterozygous for cMyBP-C truncation, show normal cMyBP-C protein levels, suggesting a different mechanism. Though rare, cases of patients homozygous for cMyBP-C truncations, present with early postnatal cardiomyopathy rapidly progressing to heart failure. We postulated that human iPSC-ECTs would present with a hypercontractile phenotype with accelerated contractile kinetics, similar to cMyBP-C -/- mouse ECTs. We introduced homozygous (-/-) and heterozygous (+/-) frameshifts in cMyBP-C in iPSCs by CRISPR gene editing followed by differentiation into CM which were incorporated into ECTs. Although cMyBP-C protein levels were similar in +/- and +/+ 2D CM, the level of cMyBP-C was reduced in +/- ECTs (0.25 vs. 0.49; p = 0.007; N ≥ 8,) and was absent in -/- ECTs. Functional characterization revealed slow Ca 2+ -handling that became more pronounced with prolonged time in culture (Ca 2+ T 100 : 110.9 vs. 76.0 ms; p = 0.0006; N ≥ 7) and normal Ca 2+ -sensitivity, without pronounced differences in contractile function in +/- ECTs. In addition to slow Ca 2+ -handling, -/- ECTs (Ca 2+ T 100 : 133.3 vs. 76.0 ms; p < 0.0001; N ≥ 8) also present with decreased Ca 2+ -transient (0.24 vs. 0.65 F 340 /F 380 ; P = 0.002; N ≥ 8) and twitch (3.93 vs. 10.30 mN/mm 2 ; p < 0.0001; N ≥ 21) amplitudes, and slow late relaxation (RT 50-90 120.0 vs. 95.1 ms; p < 0.0001; N ≥ 21) that became more pronounced with prolonged time in culture. When permeabilized, -/- ECT displayed a right-shift in Ca 2+ -sensitivity (pCa 50 5.87 vs. 5.98; p < 0.0001; N ≥ 11) and a decrease in Hill-coefficient (2.24 vs. 2.98; p = 0.0006; N ≥ 11). In our living 3D in vitro tissue culture system, heterozygous truncation of cMyBP-C causes haploinsufficiency and demonstrates a slow Ca 2+ -release with minimal contractile abnormalities, while -/- ECTs present with a hypocontractile phenotype, likely mediated by aberrant Ca 2+ handling and sensitivity. These findings reinforce that Ca 2+ -mishandling is a primary defect in cMyBP-C HCM, and therefore represents a target for therapeutical intervention.