Aims The exercise-induced beneficial mechanisms after long-term myocardial infarction (MI) are incompletely understood. The present study evaluated the effect of treadmill exercise training (5 weeks), started at a late stage of heart failure (HF) (13 weeks post-MI), on rat left ventricle remodelling and dysfunction of the regional global and cellular contractile functions. Methods and results In vivo echocardiography confirmed that sub-endocardial (ENDO) layers contract more (þ86%) and faster (þ50%) than the sub-epicardial (EPI) layers. This gradient was lost in MI rats due to a predominant reduction in the ENDO layer contractility. Exercise partially restored the amplitude and velocity of ENDO contraction, resulting in a partial recovery of the pump function indexed by the aortic blood-flow velocity time integral. At the cellular level, MI impaired ENDO contractile properties by reducing cell shortening (10–7%), calcium transient, and myofilament Ca 2þ sensitivity. These alterations were normalized by exercise. Sarcoplasmic reticulum Ca 2þ-ATPase (SERCA)2a expression and myosin light chain (MLC)-2 phosphorylation in ENDO cells were significantly reduced after MI and were restored by exercise. The EPI layer was only slightly reduced in vivo without cellular alterations. Conclusion This study shows that exercise performed at a late stage after MI restored a transmural non-uniformity of myocardium lost during HF. Recoveries of Ca 2þ homeostasis and myofilament function of cardiomyocytes contribute to this beneficial effect.