Cardiac troponin I (cTnI) is well known as a biomarker for the diagnosis of myocardial damage. However, because of its central role in the regulation of contraction and relaxation in heart muscle, cTnI may also be a potential target for the treatment of heart failure. Studies in rodent models of cardiac disease and human heart samples showed altered phosphorylation at various sites on cTnI (i.e. site-specific phosphorylation). This is caused by altered expression and/or activity of kinases and phosphatases during heart failure development. It is not known whether these (transient) alterations in cTnI phosphorylation are beneficial or detrimental. Knowledge of the effects of site-specific cTnI phosphorylation on cardiomyocyte contractility is therefore of utmost importance for the development of new therapeutic strategies in patients with heart failure. In this review we focus on the role of cTnI phosphorylation in the healthy heart upon activation of the beta-adrenergic receptor pathway (as occurs during increased stress and exercise) and as a modulator of the Frank-Starling mechanism. Moreover, we provide an overview of recent studies which aimed to reveal the functional consequences of changes in cTnI phosphorylation in cardiac disease.