The increased use of proton therapy has led to the need of better understanding the cellular mechanisms involved. The aim of this study was to investigate the effects induced by the accelerated proton beam in hepatocarcinoma cells. An existing facility in IFIN-HH, a 3 MV Tandetron™ accelerator, was used to irradiate HepG2 human hepatocarcinoma cells with doses between 0 and 3 Gy. Colony formation was used to assess the influence of radiation on cell long-term replication. Also, the changes induced at the mitochondrial level were shown by increased ROS and ATP levels as well as a decrease in the mitochondrial membrane potential. An increased dose has induced DNA damages and G₂/M cell cycle arrest which leads to caspase 3/7 mediated apoptosis and senescence induction. Finally, the morphological and ultrastructural changes were observed at the membrane level and the nucleus of the irradiated cells. Thus, proton irradiation induces both morphological and functional changes in HepG2 cells.