DNA is the major target of radiation therapy of malignant tumors. Ionizing radiation (IR) induces a variety of DNA lesions, including chemically modified bases and strand breaks. The use of proton beam therapy for cancer treatment is ramping up, as it is expected to reduce normal tissue damage. Thus, it is important to understand the molecular mechanisms of recognition, signaling, and repair of DNA damage induced by protons in the perspective of assessing not only the risk associated with human exposure to IR but also the possibility to improve the efficacy of therapy. Here, we used targeted irradiation of nuclear regions of living cells with controlled number of protons at a high spatio-temporal resolution to detect the induced base lesions and characterize the recruitment kinetics of the specific DNA glycosylases to DNA damage sites. We show that localized irradiation with 4 MeV protons induces, in addition to DNA double strand breaks (DSBs), the oxidized bases 7,8-dihydro-8-oxoguanine (8-oxoG) and thymine glycol (TG) at the site of irradiation. Consistently, the DNA glycosylases OGG1 and NTH1, capable of excising 8-oxoG and TG, respectively, and initiating the base excision repair (BER) pathway, are recruited to the site of damage. To our knowledge, this is the first direct evidence indicating that proton microbeams induce oxidative base damage, and thus implicating BER in the repair of DNA lesions induced by protons.