Objectives: This study was designed to assess whether post-myocardial infarction (MI) in-scar transplantation of skeletal myoblasts (SM) could reduce chronic ischemic mitral regurgitation (MR) by decreasing left ventricular (LV) remodeling. Background: Extensive work has confirmed the relationship between ischemic MR and post-myocardial infarction (MI) remodeling of the LV. Methods: An infero-posterior MI was created in 13 sheep, thereby resulting in increasing MR. Two months post-MI, the animals were randomized and in-scar injected with expanded autologous SM (n = 6, mean: 251 × 106 cells) or culture medium only (n = 7). Three-dimensional echocardiography was performed at baseline, before transplantation, and for two months thereafter (sacrifice), with measurements of LV end-diastolic and end-systolic volumes (ESV), ejection fraction (EF), MR stroke volume, and leaflet tethering distance; wall motion score index (WMSi) was assessed by two-dimensional echo. Results: Measurements were similar between groups at baseline and before transplantation. At sacrifice, transplantation was found to have reduced MR progression (regurgitant volume change: -1.83 ± 0.32 ml vs. 5.9 ± 0.7 ml in control group, p < 0.0001) and tethering distance (-0.41 ± 0.09 cm vs. 0.44 ± 0.12 cm in control group, p < 0.001), with significant improvement of EF (2.01 ± 0.94% vs. -4.86 ± 2.23%, p = 0.02), WMSi (-0.25 ± 0.11 vs. 0.13 ± 0.03 in controls, p < 0.01) and a trend to a lesser increase in ESV (23.3 ± 3.5 ml vs. 35.4 ± 4.2 ml in control group, p = 0.055). Conclusions: Autologous skeletal myoblast transplantation attenuates mild-to-moderate chronic ischemic MR, which otherwise is progressive, by decreasing tethering distance and improving EF and wall motion score, thereby enhancing valve coaptation. These data shed additional light on the mechanism by which skeletal myoblast transplantation may be cardioprotective. © 2006 American College of Cardiology Foundation.