In the current study, the lethality as well as the immunological, biochemical and histological effects of Naja nigricollis (Black-necked spitting cobra) venom at a sublethal dose has been investigated before and after exposure to gamma radiation (1.5KGy and 3KGy). Data revealed that the toxicity of irradiated venom (1.5KGy & 3KGy) decreased as compared to that of the native one. The LD50 for native and gamma-irradiated (1.5KGy & 3KGy) Naja nigricollis venom was 0.440 mg/kg, 4.79 mg/kg and 5.38 mg/kg, respectively. Irradiation of the whole venom with (1.5kGy & 3kGy) reduced its lethality 10.8 and 12.22 times as compared to its native venom, respectively. The immunodiffusion technique showed same number of precipitin bands against polyvalent antivenin with the native and the two dose levels gamma-irradiated venoms. There was no change in the antigenic reactivity between both native and irradiated Naja nigricollis snake venom. As for the biochemical responses of Naja nigricollis venom, the effect of half LD50 of native or irradiated (1.5KGy) was studied on the activities of heart enzymes: CPK, CK-MB, LDH and AST after (1, 2, 4, 24 hours) of envenomation. The present study showed that snake venom envenomation caused significant (p ≤ 0.05) elevation in serum CPK, CK-MB, LDH and AST levels. In contrast, the 1.5KGy gamma-irradiated Naja nigricollis venom recorded no significant changes compared to that of the non envenomated normal rats. These results are in accordance with the histological findings of the heart that showed severe degeneration of the cardiac muscle with loss of striations and extensive haemorrhage inbetween the myocardial bundles. These results indicate that 1.5KGy gamma irradiation of venoms offer an effective method for reducing the chronic toxic effect of venom in immunized animals for preparing the best toxoids and vaccines, facilitating antisera production and extending the useful life of immunized animals.