Introduction: Mutations in MYPBC3, the gene encoding cardiac Myosin-Binding Protein C (cMyBP-C), a regulatory sarcomeric protein, are known to play a pathogenic role in hypertrophic cardiomyopathy (HCM). However, the molecular bases for this pathophysiology remain poorly understood. Here, a novel large-animal MYPBC3-mutant model was developed and characterized to determine the association between myofilament abnormalities induced by cMyBP-C dysfunction and the resulting in vivo phenotype. Methods: A missense (early stop) E330X MYPBC3 mutation was introduced in Yucatan mini-pigs. Young heterozygous mutants (C3) and age-matched wildtype (WT) herd-mates were studied. In vivo imaging (MRI) and invasive hemodynamics were performed along with ex vivo measurements of tension dynamics, sarcomere structure (x-ray diffraction), and ATP turnover rates, including the population of myosin heads in the super-relaxed state (SRX) in LV fibers/tissues. Load-independent systolic and diastolic function (via LV pressure-volume relationships), and β-adrenergic receptor (β-AR) cardiac reserve (dobutamine 5 ug/kg/min IV) were assessed in vivo. Results: Relaxed C3 fibers showed reduced ordering of the myosin heads around the thick filament and a sensitizing shift of myosin-heads towards actin. However, C3 pigs showed normal Ca2+ sensitivity ex vivo with mildly decreased systolic function at rest in vivo (LVEF: 49 ± 1 vs. 56 ± 2%; vmax: 31 ± 1 vs. 38 ± 5 1/s; Ees: 8.5 ± 1.0 vs. 10.2 ± 1.5 mmHg/mL; all P < 0.05 vs. CTRL). Upon β-AR stimulation, C3 pigs became hypercontractile (Ees: 18.0 ± 3.1 vs. 14.3 ± 2.8 mmHg/mL in CTRL, P <0.05) showing markedly increased (P<0.05) end-diastolic stiffness (Eed: 1.7 ± 0.4 vs. 1.3 ± 0.3 mmHg/mL in CTRL) and pressures; C3 fibers showed SRX dysregulation dependent on the phosphorylation state. Conclusions: MYPBC3 mutant mini-pigs showed a complex phenotype, characterized by myofilament abnormalities ex vivo and β-AR dependent hypercontractility and diastolic dysfunction in vivo . Taken together, these data generated in a translationally-relevant novel large-animal model further support the role of cMyBP-C as a key regulator of contraction and relaxation, but also can provide insights into the pathophysiology of HCM.