Immune system is amongst the most radiosensitive system to radiation-induced cellular and molecular damage. Present study was focused on the evaluation of radioprotective efficacy of a novel secondary metabolite, N-acetyl tryptophan glucoside (NATG), isolated from a radioresistant bacterium Bacillus sp. INM-1 using murine macrophage J774A.1 cells experimental model. Radioprotective efficacy of NATG against radiation-induced DNA damage and apoptosis was estimated using phosphatidyl-serine-externalization Annexin V-PI and Comet assay analysis. Radiation-induced cell death is the outcome of oxidative stress caused by free radicals. Therefore, perturbations in antioxidant enzymes i.e., superoxide dismutase (SOD), catalase, glutathione-s-transferase (GST) and GSH activities in irradiated and NATG pre-treated irradiated J774A.1 cells were studied. Results of the present study demonstrated that NATG pre-treated (0.25 µg/ml) irradiated (20 Gy) cells showed significant (p < 0.05) reduction in apoptotic cells index at 4-48 h as compared to radiation alone cells. Comet assay exhibited significant protection to radiation-induced DNA damage in J774A.1 cells. Significantly shortened DNA tail length, increased % Head DNA contents and lower olive tail moment was observed in NATG pre-treated irradiated cells as compared to radiation alone cells. Further, significant increase in catalase (~ 3.9 fold), SOD (67.52%), GST (~ 1.9 fold), and GSH (~ 2.5 fold) levels was observed in irradiated cells pre-treated with NATG as compared to radiation-alone cells. In conclusion, current study suggested that NATG pre-treatment to irradiated cells enhanced antioxidant enzymes in cellular milieu that may contribute to reduce oxidative stress and decrease DNA damage which resulted to significant reduction in the cell death of irradiated macrophages.