Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca²⁺-dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two Ca²⁺ bound state) in complex with the N-terminal fragment of TnI (TnI_1–47). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R factor of 22.2% (R_free = 32.6%). The central, connecting α-helix observed in the structure of uncomplexed TnC (TnC_free) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90°. As a result, TnC in the complex has a compact globular shape with direct interactions between the N- and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI_1–47 α-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the TnI_1–47 α-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between Ca²⁺ receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC/TnI_1–47 structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique α-helical regulatory TnI segment, which binds to the N-lobe of TnC in its Ca²⁺ bound conformation.