•  An important mechanism in beat‐to‐beat optimization of heart performance is matching ventricular output with end‐diastolic volume, which is known as the Frank–Starling Relationship. •  The cellular basis for this regulation involves myofilament length–tension relationships. •  We previously showed two populations of length–tension relationships in mammalian left ventricular cardiac myocytes, one steep like fast‐twitch skeletal muscle fibres and the other shallow like slow‐twitch skeletal muscle fibres, and cardiac myocytes with shallow length–tension relationships shift to steep relationships by protein kinase A‐induced myofilament phosphorylation. •  The current study investigated the molecular and amino acid residue mechanisms that control length–tension relationships. •  The single muscle cell experiments demonstrated that cardiac troponin I phosphorylation at serines 23/24 control length–tension relationships in striated muscle. •  This study provides: (i) a mechanism to explain a length dependence of force generation in striated muscle; and (ii) an important target to potentially treat heart disease associated with compromised Frank–Starling relationships. Abstract  According to the Frank–Starling relationship, greater end‐diastolic volume increases ventricular output. The Frank–Starling relationship is based, in part, on the length–tension relationship in cardiac myocytes. Recently, we identified a dichotomy in the steepness of length–tension relationships in mammalian cardiac myocytes that was dependent upon protein kinase A (PKA)‐induced myofibrillar phosphorylation. Because PKA has multiple myofibrillar substrates including titin, myosin‐binding protein‐C and cardiac troponin I (cTnI), we sought to define if phosphorylation of one of these molecules could control length–tension relationships. We focused on cTnI as troponin can be exchanged in permeabilized striated muscle cell preparations, and tested the hypothesis that phosphorylation of cTnI modulates length dependence of force generation. For these experiments, we exchanged unphosphorylated recombinant cTn into either a rat cardiac myocyte preparation or a skinned slow‐twitch skeletal muscle fibre. In all cases unphosphorylated cTn yielded a shallow length–tension relationship, which was shifted to a steep relationship after PKA treatment. Furthermore, exchange with cTn having cTnI serines 23/24 mutated to aspartic acids to mimic phosphorylation always shifted a shallow length–tension relationship to a steep relationship. Overall, these results indicate that phosphorylation of cTnI serines 23/24 is a key regulator of length dependence of force generation in striated muscle.