Cardiac troponin I (cTnI) is a phosphoprotein subunit of the troponin‐tropomyosin complex that is thought to inhibit cardiac muscle contraction during diastole. To investigate the contributions of cTnI phosphorylation to cardiac regulation, transgenic mice were created with the phosphorylation sites of cTnI mutated to alanine. Activation of protein kinase C (PKC) by perfusion of hearts with phorbol‐12‐myristate‐13‐acetate (PMA) or endothelin‐1 (ET‐1) inhibited the maximum ATPase rate by up to 25 % and increased the Ca²⁺ sensitivity of ATPase activity and of isometric tension by up to 0.15 pCa units. PKC activation no longer altered cTnI phosphorylation, depressed ATPase rates or enhanced myofilament Ca²⁺ sensitivity in transgenic mice expressing cTnI that could not be phosphorylated on serines^43/45 and threonine¹⁴⁴ (PKC sites). Modest changes in myosin regulatory light chain phosphorylation occurred in all mouse lines, but increases in myofilament Ca²⁺ sensitivity required the presence of phosphorylatable cTnI. For comparison, the β‐adrenergic agonist isoproterenol caused a 38 % increase in maximum ATPase rate and a 0.12 pCa unit decrease in myofilament Ca²⁺ sensitivity. These β‐adrenergic effects were absent in transgenic mice expressing cTnI that could not be phosphorylated on serines^23/24 (protein kinase A, PKA, sites). Overall, the results indicate that PKC and PKA exert opposing effects on actomyosin function by phosphorylating cTnI on distinct sites. A primary role of PKC phosphorylation of cTnI may be to reduce the requirements of the contractile apparatus for both Ca²⁺ and ATP, thereby promoting efficient ATP utilisation during contraction.