Protection of animals immunized against Bacillus anthracis is usually demonstrated by challenging with an appropriate dose of a strain of Bacillus anthracis that is lethal to unvaccinated animals inoculated at the same time. In this study the protective efficacy in anthrax vaccine (34F₂ sterne strain spore) was evaluated in sheep, goats and guinea pigs challenged with subcutaneous inoculation of a highly virulent Bacillus anthracis spores recently isolated from soil in Iran. This strain has been shown that it is lethal for sheep, goats and guinea pigs in very low dose. It was found that the vaccine provides highly protection (100 % ) after challenging the immunized animals against this strain in sheep, goats and guinea pigs. So, indicates the superior protective efficacy of the vaccine. The authors recommend that this strain can be used instead of other strain which has been used previously for challenging the immunized animals, and also guinea pig is a good substitution for evaluating anthrax vaccine.