Moderate physical activity has traditionally been associated with the improvement of cardiac function and, consequently, with the extension of life span. Mitochondria play a key role in the adaptation of heart muscle to exercise-related metabolic demands. In order to disclose the molecular mechanisms underlying the beneficial effect of lifelong physical activity in cardiac function, we performed label-free quantitative mass spectrometry-based proteomics of Sprague-Dawley rat heart mitochondrial proteome and phosphoproteome. Our data revealed that 54 weeks of moderate treadmill exercise modulates the abundance of proteins involved in the generation of precursor metabolites and cellular respiration, suggesting an increase in carbohydrate oxidation-based metabolism. Moreover, from the 1335 phosphopeptides identified in this study, 6 phosphosites were exclusively assigned to heart mitochondria from sedentary rats and 17 to exercised animals, corresponding to 6 and 16 proteins, respectively. Most proteins exhibiting significant alterations in specific phosphorylation sites were involved in metabolism. Analysis of the acquired data led to the identification of several kinases potentially modulated by exercise training, which were selected for further validation. Indeed, higher protein abundance levels of RAF and p38 in mitochondria were confirmed to be modulated by sustained exercise. Our work describes the plasticity of heart mitochondria in response to long exercise programs manifested by the reprogramming of phosphoproteome and provides evidence for the kinases involved in the regulation of metabolic pathways and mitochondrial maintenance.