DNA integrity and stability are vital for proper cellular activity. Nevertheless, to treat cancer patients, DNA is the main target for inducing tumoral cell death. Nowadays, cancer treatment is improving by the development of new technologies, protocols and strategies. Amongst them, the introduction of charged particles as a form of radiotherapy is underway. However, tumor-neighboring healthy tissues are still exposed to ionizing radiations (IR) and subject to late side effects. Skeletal muscle is one of those tissues most likely to be affected. To decipher the DNA damage response (DDR) of skeletal muscle cells, myogenic cells, we irradiated them with microbeams of protons or α-particles and followed the accumulation of DDR proteins at irradiation sites. Thereby, we showed that myoblasts, proliferating myogenic cells, repair IR-induced DNA damage through both non-homologous end-joining and homologous recombination with different recruitment dynamics depending on the characteristics of ionizing particles (type, energy deposition and time after irradiation), whereas myotubes, post-mitotic myogenic cells, display reduced DNA damage response.SummaryMouse skeletal muscle cells and their precursor cells irradiated by ion microbeam show DNA damage response and DNA repair dynamics that vary according to the characteristics of ionizing particles delivered.Graphical Abstract