β-adrenergic stimulation plays a critical role in accelerating ventricular contraction and speeding relaxation to match cardiac output to changing circulatory demands. Two key myofilaments proteins, troponin I (TnI) and myosin binding protein-C (MyBP-C), are phosphorylated following β-adrenergic stimulation; however, their relative contributions to the enhancement of in vivo cardiac contractility are unknown. To examine the roles of TnI and MyBP-C phosphorylation in β-adrenergic-mediated enhancement of cardiac function, transgenic (TG) mice expressing non-phosphorylatable TnI protein kinase A (PKA) residues (i.e. serine to alanine substitution at Ser23/24; TnIPKA−) were bred with mice expressing non-phosphorylatable MyBP-C PKA residues (i.e. serine to alanine substitution at Ser273, Ser282 and Ser302; MyBPCPKA−) to generate a novel mouse model expressing non-phosphorylatable PKA residues in TnI and MyBP-C (DBLPKA−). MyBP-C dephosphorylation produced cardiac hypertrophy and increased wall thickness in MyBPCPKA− and DBLPKA− mice, and in vivo echocardiography and pressure–volume catheterization studies revealed impaired systolic function and prolonged diastolic relaxation compared to wild-type and TnIPKA– mice. Infusion of the β-agonist dobutamine resulted in accelerated rates of pressure development and relaxation in all mice; however, MyBPCPKA− and DBLPKA− mice displayed a blunted contractile response compared to wild-type and TnIPKA– mice. Furthermore, unanaesthesized MyBPCPKA− and DBLPKA− mice displayed depressed maximum systolic pressure in response to dobutamine as measured using implantable telemetry devices. Taken together, our data show that MyBP-C phosphorylation is a critical modulator of the in vivo acceleration of pressure development and relaxation as a result of enhanced β-adrenergic stimulation, and reduced MyBP-C phosphorylation may underlie depressed adrenergic reserve in heart failure.