Traditionally, it was thought that exercise improved cardiac function by only increasing myocardial mass and contractility through physiological hypertrophy of existing myocytes. Recent data on myocardial cell homeostasis and the identification of cardiac stem cells (CSCs), in the adult mammalian heart, could challenge this concept. We sought to assess if CSC activation and ensuing myocyte formation participates in cardiac remodeling induced by exercise. To this aim, 36 FVB mice underwent a programme of controlled swimming exercise (Ex) and were sacrificed at different time points over 28 days. Ten untrained mice acted as sedentary controls (Sed). To track myocardial proliferation, BrdU was administered (i.p.) twice daily. Hearts were processed for immunohistochemistry and confocal microscopy analysis (n=30) and c-kit-positive CSCs and myocytes were also isolated for molecular analyses (n=16). Results showed that exercise training resulted in increased heart:body weight ratios in Ex (6.1±0.7mg/g, Mean±SD) vs. Sed (4.3±0.2mg/g) mice. Average myocyte volume was greater in Ex mice, compared to Sed. The distribution of myocyte sizes showed the presence of both larger (hypertrophied) and smaller myocytes in the ventricular wall of the Ex vs. Sed hearts. To address the source of these smaller myocytes, we evaluated CSC myocardial activation. The number of CSCs increased 5-fold in the ventricular wall of the Ex vs. Sed mice. Most CSCs were proliferating (BrdU and/or Ki-67 positive) and many expressed the cardiac transcription factor, GATA-4, representing cardiac progenitors. Small amounts of sarcomeric proteins were found in the cytoplasm of some of these cells, corresponding to myocyte precursors. Consequently, we detected increased numbers of small BrdU (3±1%) and Ki-67 (1.3±0.1%) positive myocytes in Ex vs. Sed (BrdU=0.03±0.01%;Ki-67=0.01±0.01%) hearts. These cells were transiently cell cycle competent as shown by their expression of p107 instead of pRB, followed by E2F1 and c-myc. Also, Cyclin E/cdk2 and Cyclin B1/cdc2 complexes were increased in the small myocytes isolated from Ex mice. IGF-1 expression progressively increased in myocytes, and then in CSCs, over 28 days of exercise training. This increased IGF-1 expression resulted in a higher number of CSCs, through IGF-1R activation and Akt phosphorylation in CSCs, isolated from Ex compared to Sed mice. This demonstrates that the IGF-1Rs are functional and signal to their downstream physiological targets in the activated CSCs. In conclusion, exercise training results in myocardial mass remodeling through both myocyte hypertrophy as well as hyperplasia. The latter is due to the activation and ensuing differentiation of CSCs into newly-formed myocytes, with increased IGF-1 expression playing a key role in this process.