Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca2+ levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca2+ concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow). These effects were greater for submaximal Ca 2+ activated contractions. PKA treatment also reduced the rate of contractile activation (kACT) at maximal, but not submaximal Ca 2+, and reduced the Ca2+ sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca2+-cTn binding affinity and C-I interaction were monitored. Ca2+ binding to cTn (pCa50) was significantly decreased when cTnI was phosphorylated by PKA (ΔpCa50 = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca2+. These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during β-adrenergic stimulation. © 2014 Biophysical Society.