Gamma radiation‐induced cell death is associated with and mediated by enzymatic DNA fragmentation. A nuclease that produces this fragmentation of DNA is unknown. To determine whether deoxyribonuclease I (DNase I), a major cytotoxic/apoptotic nuclease expressed in all cells and tissues, can mediate gamma radiation‐induced tissue injury, we used DNase I knockout (KO) mice and a zinc chelate of 3,5‐diisopropylsalicylic acid (Zn‐DIPS) that, as we show, has DNase I inhibiting activity in vitro. Our study demonstrated that inactivation or inhibition of DNase I strongly suppressed radiation injury (8 Gy, LD50/30) to white pulp of spleen, intestine villi, and bone marrow as measured using quantitative TUNEL assay. Spleen and intestine of DNase I KO mice were additionally protected from radiation by Zn‐DIPS due to the broad radioprotective efficacy of zinc. It is interesting that main DNase I‐producing tissues such as salivary gland, pancreas, and kidney showed no effect of DNase I inactivation. These results suggest that DNase I‐mediated mechanism of DNA damage and tissue injury has important regulatory role in radiation‐mediated cell death in radiosensitive organs. The study was supported by NIH/NIDDK and VA Merit Review grants to A.G.B.