Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.