this work was conducted to study the inactivation dynamics of Rabies Virus (RV) by calcinated eggshell calcium oxide (CaO) in comparison with Binary Ethyleneimine (BEI). Calcinated eggshell, of which the main component is calcium oxide, was evaluated in the form of powder as inactivator to rabies virus. The calcination process yielded calcium oxide with a purity of 97.92 % w/w. The results of X-R diffraction showed that the optimum calcination conditions for preparation of CaO from chicken eggshell is 900ºC for 1 hour with peak 2ɵ (2-Theta) at 32.3º, 37.4°, 54.0º, 64.3°and 67.5º. Complete virus inactivation was obtained with BEI (3% of 0.001M) after incubation for 3 hours. On the other hand, 10% Egg-CaO solution inactivated RV within 15 minutes. The safety and potency of CaO prepared inactivated rabies vaccines were evaluated in three groups of puppies. The inactivated rabies vaccine produced sufficient level of antibodies as measured by serum neutralization test (SNT) and was protected when challenged in mice. The obtained results revealed that no residual infectious virus was detected in Egg-CaO inactivated rabies vaccine that proved to be safe and effective as compared the same virus that inactivated with the classical inactivating agent.