It has been reported that DNA double-strand breaks (DSB) can be induced by cytoplasm irradiation, and that both reactive free radicals and mitochondria are involved in DSB formation. However, the cellular antioxidative responses that are stimulated and the biological consequences of cytoplasmic irradiation remain unknown. Using the Single Particle Irradiation system to Cell (SPICE) proton microbeam facility at the National Institute of Radiological Sciences ([NIRS] Japan), the response of nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidative signaling to cytoplasmic irradiation was studied in normal human lung fibroblast WI-38 cells. Cytoplasmic irradiation stimulated the localization of NRF2 to the nucleus and the expression of its target protein, heme oxygenase 1. Activation of NRF2 by tert-butylhydroquinone mitigated the levels of DSB induced by cytoplasmic irradiation. Mitochondrial fragmentation was also promoted by cytoplasmic irradiation, and treatment with mitochondrial division inhibitor 1 (Mdivi-1) suppressed cytoplasmic irradiation-induced NRF2 activation and aggravated DSB formation. Furthermore, p53 contributed to the induction of mitochondrial fragmentation and activation of NRF2, although the expression of p53 was significantly downregulated by cytoplasmic irradiation. Finally, mitochondrial superoxide (MitoSOX) production was enhanced under cytoplasmic irradiation, and use of the MitoSOX scavenger mitoTEMPOL indicated that MitoSOX caused alterations in p53 expression, mitochondrial dynamics, and NRF2 activation. Overall, NRF2 antioxidative response is suggested to play a key role against genomic DNA damage under cytoplasmic irradiation. Additionally, the upstream regulators of NRF2 provide new clues on cytoplasmic irradiation-induced biological processes and prevention of radiation risks.