Cardiac troponin I (cTnI) phosphorylation modulates myocardial contractility and relaxation during -adrenergic stimulation. cTnI differs from the skeletal isoform in that it has a cardiac specific N` extension of 32 residues (N` extension). The role of the acidic N` region in modulating cardiac contractility has not been fully defined. To test the hypothesis that the acidic N` region of cTnI helps regulate myocardial function, we generated cardiac-specific transgenic mice in which residues 2–11 (cTnI^2–11) were deleted. The hearts displayed significantly decreased contraction and relaxation under basal and -adrenergic stress compared to nontransgenic hearts, with a reduction in maximal Ca²⁺-dependent force and maximal Ca²⁺-activated Mg²⁺-ATPase activity. However, Ca²⁺ sensitivity of force development and cTnI-Ser^23/24 phosphorylation were not affected. Chemical shift mapping shows that both cTnI and cTnI^2–11 interact with the N lobe of cardiac troponin C (cTnC) and that phosphorylation at Ser^23/24 weakens these interactions. These observations suggest that residues 2–11 of cTnI, comprising the acidic N` region, do not play a direct role in the calcium-induced transition in the cardiac regulatory or N lobe of cTnC. We hypothesized that phosphorylation at Ser<sup>23/24</sup> induces a large conformational change positioning the conserved acidic N region to compete with actin for the inhibitory region of cTnI. Consistent with this hypothesis, deletion of the conserved acidic N` region results in a decrease in myocardial contractility in the cTnI^2–11 mice demonstrating the importance of acidic N` region in regulating myocardial contractility and mediating the response of the heart to -AR stimulation.