Myosin-binding protein-C (MyBP-C) is a thick filament-associated protein that binds tightly to myosin. Given that cMyBP-C may act to modulate cooperative activation of the thin filament by constraining the availability of myosin cross-bridges for binding to actin, we investigated the role of MyBP-C in the regulation of cardiac muscle contraction. We assessed the Ca²⁺ sensitivity of force (pCa₅₀) and the activation dependence of the rate of force redevelopment (k_tr) in skinned myocardium isolated from wild-type (WT) and cMyBP-C null (cMyBP-C^−/−) mice. Mechanical measurements were performed at 22°C in the absence and presence of a strong-binding, nonforce-generating analog of myosin subfragment-1 (NEM-S1). In the absence of NEM-S1, maximal force and k_tr and the pCa₅₀ of isometric force did not differ between WT and cMyBP-C^−/− myocardium; however, ablation of cMyBP-C-accelerated k_tr at each submaximal force. Treatment of WT and cMyBP-C^−/− myocardium with 3μM NEM-S1 elicited similar increases in pCa_50, but the effects of NEM-S1 to increase k_tr at submaximal forces and thereby markedly reduce the activation dependence of k_tr occurred to a greater degree in cMyBP-C^−/− myocardium. Together, these results support the idea that cMyBP-C normally acts to constrain the interaction between myosin and actin, which in turn limits steady-state force development and the kinetics of cross-bridge interaction.