cTnI(P82S) (cTnI(P83S) in rodents) resides at the I-T arm of cardiac troponin I (cTnI) and was initially identified as a disease-causing mutation of hypertrophic cardiomyopathy (HCM). However, later studies suggested this may not be true. We recently reported that introduction of an HCM-associated mutation in either inhibitory-peptide (cTnI(R146G)) or cardiac specific N-terminus (cTnI(P21C)) of cTnI blunts the PKA-mediated modulation on myofibril activation/relaxation kinetics by prohibiting formation of intrasubunit contacts between these regions. Here, we tested whether this also occurs for cTnI(P83S). cTnI(P83S) increased both Ca2+ binding affinity to cTn (K-ca) and affinity of cTnC for cTnI (KC-I), and eliminated the reduction of K-ca and KC-I observed for phosphorylated-cTnI(WT). In isolated myofibrils, cTnI(P83S) maintained maximal tension (T-MAX) and Ca2+ sensitivity of tension (pCa(50)). For cTnI(WT) myofibrils, PKA-mediated phosphorylation decreased pCa(50) and sped up the slow-phase relaxation (especially for those Ca2+ conditions that heart performs in vivo). Those effects were blunted for cTnI(P83S) myofibrils. Molecular-dynamics simulations suggested cTnI(P83S) moderately inhibited an intrasubunit interaction formation between inhibitory-peptide and N-terminus, but this "blunting" effect was weaker than that with cTnI(R146G) or cTnI(P21C). In summary, cTnI(P83S) has similar effects as other HCM-associated cTnI mutations on troponin and myofibril function even though it is in the I-T arm of cTnI.