Phosphorylation of cardiac myosin binding protein-C (MyBP-C) modulates cardiac contractile function; however, the specific roles of individual serines (Ser) within the M-domain that are targets for β-adrenergic signaling are not known. Recently, we demonstrated that significant accelerations in in vivo pressure development following β-agonist infusion can occur in transgenic (TG) mouse hearts expressing phospho-ablated Ser²⁸² (that is, TG^S282A) but not in hearts expressing phospho-ablation of all three serines [that is, Ser²⁷³, Ser²⁸², and Ser³⁰² (TG^3SA)], suggesting an important modulatory role for other Ser residues. In this regard, there is evidence that Ser³⁰² phosphorylation may be a key contributor to the β-agonist–induced positive inotropic responses in the myocardium, but its precise functional role has not been established. Thus, to determine the in vivo and in vitro functional roles of Ser³⁰² phosphorylation, we generated TG mice expressing nonphosphorylatable Ser³⁰² (that is, TG^S302A). Left ventricular pressure-volume measurements revealed that TG^S302A mice displayed no accelerations in the rate of systolic pressure rise and an inability to maintain systolic pressure following dobutamine infusion similar to TG^3SA mice, implicating Ser³⁰² phosphorylation as a critical regulator of enhanced systolic performance during β-adrenergic stress. Dynamic strain–induced cross-bridge (XB) measurements in skinned myocardium isolated from TG^S302A hearts showed that the molecular basis for impaired β-adrenergic–mediated enhancements in systolic function is due to the absence of protein kinase A–mediated accelerations in the rate of cooperative XB recruitment. These results demonstrate that Ser³⁰² phosphorylation regulates cardiac contractile reserve by enhancing contractile responses during β-adrenergic stress.