The objective of this work was to investigate the role of acidic residues within the exposed middle segment of the central helix of cTnC in (1) cTnC-cTnI interactions, (2) Ca2+ binding and exchange with the regulatory N-domain of cTnC in increasingly complex biochemical systems, and (3) ability of the cTn complex to regulate actomyosin ATPase. In order to achieve this objective, we introduced the D87A/D88A and E94A/E95A/E96A mutations into the central helix of cTnC. The D87A/D88A and E94A/E95A/E96A mutations decreased affinity of cTnC for the regulatory region of cTnI. The Ca2+ sensitivity of the regulatory N-domain of isolated cTnC was decreased by the D87A/D88A, but not E94A/E95A/E96A mutation. However, both the D87A/D88A and E94A/E95A/E96A mutations desensitized the cTn complex and reconstituted thin filaments to Ca2+. Decreases in the Ca2+ sensitivity of the cTn complex and reconstituted thin filaments were, at least in part, due to faster rates of Ca2+ dissociation. In addition, the D87A/D88A and E94A/E95A/E96A mutations desensitized actomyosin ATPase to Ca2+, and decreased maximal actomyosin ATPase activity. Thus, our results indicate that conserved acidic residues within the exposed middle segment of the central helix of cTnC are important for the proper regulatory function of the cTn complex.