Ionizing radiation increases cell mortality in a dose-dependent manner. Increases in DNA double strand breaks, γ-H2AX, p53 phophorylation, and protein levels of p53 and Bax also occur. We investigated the ability of ciprofloxacin (CIP), a widely prescribed antibiotic, to inhibit DNA damage induced by ionizing radiation. Human tumor TK6, NH32 (p53 (-/-) of TK6) cells, and human normal peripheral blood mononuclear cells (PBMCs) were exposed to 2-8 Gy (60)Co-γ-photon radiation. γ-H2AX (an indicator of DNA strand breaks), phosphorylated p53 (responsible for cell-cycle arrest), Bcl-2 (an apoptotic protein, and cell death were measured. Ionizing irradiation increased γ-H2AX amounts in TK6 cells (p53(+/+)) within 1 h in a radiation dose-dependent manner. CIP pretreatment and posttreatment effectively inhibited the increase in γ-H2AX. CIP pretreatment reduced Bcl-2 production but promoted p53 phosphorylation, caspase-3 activation and cell death. In NH32 cells, CIP failed to significantly inhibit the radiation-induced γ-H2AX increase, suggesting that CIP inhibition involves in p53-dependent mechanisms. In normal healthy human PBMCs, CIP failed to block the radiation-induced γ-H2AX increase but effectively increased Bcl-2 production, but blocked the phospho-p53 increase and subsequent cell death. CIP increased Gadd45α, and enhanced p21 protein 24 h postirradiation. Results suggest that CIP exerts its effect in TK6 cells by promoting p53 phosphorylation and inhibiting Bcl-2 production and in PBMCs by inhibiting p53 phosphorylation and increasing Bcl-2 production. Our data are the first to support the view that CIP may be effective to protect normal tissue cells from radiation injury, while enhancing cancer cell death in radiation therapy.