Tumor radiotherapy with large-field irradiation results in an increase of p53-dependent apoptosis of the radiosensitive hematopoietic stem cells. Proapoptotic PUMA is a transcriptional target of p53. Thus suppression of PUMA expression by gene therapy with the transcription repressor SNAI2 as transgene might be a potential approach for normal tissue protection during radiotherapy. SNAI2 cDNA was cloned in a lentiviral SIN vector in a bicistronic expression cassette followed by a floxed IRES-EMCV linker and EGFP as selection gene. Wild-type p53 TK6 cells were used as the cellular model system. We could demonstrate the significant radioprotective effect of SNAI2 overexpression in a cytotoxicity assay after irradiation with 0–5 Gy compared with untransduced or control vector (inverse oriented SNAI2 cDNA)-transduced cells. Additionally, TK6-SNAI2 compared to TK6-SNAI2inv cells showed a survival advantage in a clonogenic assay after irradiation with 0–3 Gy. Determination of the proportion of sub-G₁ cells in TK6-SNAI2 cells revealed an approximately 50% reduction in apoptosis compared with both control entities. In this study using a bicistronic lentiviral vector, we were able to provide proof of principle that lentiviral overexpression of SNAI2 might be used for radioprotective gene therapy to widen the therapeutic range in radiotherapy.