Introduction Ionizing radiation (IR) affects healthy tissues during the treatment of cancer radiation therapy and other nuclear and radiological accidents. Some natural compounds showed nonspecific radioprotective activity with severe side effects. The present study is aimed to develop potent and specific radioprotective short hairpin RNA (shRNA), which selectively protects normal cells from IR by specifically targeting matrix metalloproteinases (MMP‐2). Results IR reduced the viability of human normal dermal fibroblasts (HDFs) in a dose‐response manner. It enhanced the expression of MMP‐2 at 10 Gy. Plasmid MMP‐2shRNA (pMMP‐2) reduced the IR (10 Gy) induced cytotoxicity analyzed by lactate dehydrogenase (LDH) assay, normalized IR induced cellular and morphological changes with enhanced the clonogenicity in 48 hours at 2 µg/mL. It reduced the ROS generation, released HDFs from G2/M arrest and rescued from apoptosis analyzed by DCFDA dye, cell cycle analysis by PI stain and annexin V assay, respectively. pMMP‐2 also modulates the expression of EGFR and reduced IR induced expression of DNA damage response protein, ATM and increased the expression of repair proteins, KU70/KU80, and RAD51. In addition, decreased the expression of cell cycle regulatory proteins cyclin‐dependent kinases (CDK1) and Cyclin B as well as proapoptotic proteins BAX, caspase‐3, and Cytochrome‐C and increased the expression of survival protein, Bcl‐2. In contrary pMMP‐2 decreased the LDH activity, survival fraction and blocked G2/M phase of cell cycle and increased apoptosis in MCF‐7 cells. In addition, decreased the expression of EGFR, proapoptotic BAX and DNA repair proteins ATM, KU70/80 and RAD51, increased expression of cyclinB as well as CDK1. Conclusion Results conclude that pMMP‐2 protected HDFs from IR and sensitized the MCF‐7 cells. Therefore, pMMP‐2 can be employed for better treatment of radiation accidents and during the treatment of radiotherapy.