Autologous mesenchymal stem cells (MSCs) have been shown to improve the functional outcome after skeletal muscle trauma. The mechanisms behind this improvement have to be answered prior to a future clinical application. We investigated for the first time the in vivo distribution and behavior of MSCs after local transplantation into a severely injured muscle with magnetic resonance imaging (MRI). Autologous rat MSCs were labeled with very small iron oxide nanoparticles (VSOPs) and transplanted into the soleus muscle 1 week after an open crush injury. Distribution and migration of the cells were evaluated in vivo over time by the repeated performance of high-resolution MRI at 7 T. Three and 6 weeks after transplantation, the muscles were histologically analyzed. The labeled MSCs could be visualized inside the traumatized muscles 24 h after transplantation showing characteristic signal reductions in T2*-weighted sequences. The hypointense signal could be followed over 6 weeks and could be easily discriminated from the structures of the injured muscle. The cell pools did not migrate inside the muscle and showed a decrease in volume over time. Prussian blue-stained histologic sections showed a topographical correlation of the respective MRI signal and nanoparticle-labeled cells. Fusion events of marked cells with regenerating myofibers could be observed. The presented study demonstrates for the first time the feasibility of an in vivo tracking of MSCs with MRI after a severe skeletal muscle injury. The investigated method can be a powerful tool both in experimental setups and in possible clinical applications of stem cell-supported skeletal muscle regeneration.