Pulmonary anthrax caused by exposure to inhaled , the most lethal form of anthrax disease, is a continued military and public health concern for the United States. The vaccine AV7909, consisting of the licensed anthrax drug substance AVA adjuvanted with CpG7909, induces high levels of toxin neutralizing antibodies in healthy adults using fewer doses than AVA. This study compares the ability of one- or two-dose regimens of AV7909 to induce a protective immune response in guinea pigs challenged with a lethal dose of aerosolized spores 6 weeks after the last vaccine dose. The results indicated that AV7909 was less effective when delivered as a single dose compared to the two-dose regimen that resulted in dose-dependent protection against death. The toxin neutralizing assay (TNA) titer and anti-PA IgG responses were proportional to the protective efficacy, with a 50% TNA neutralizing factor (NF ) greater than 0.1 associated with survival in animals receiving two doses of vaccine. The strong protection at relatively low TNA NF titers in this guinea pig model supports the exploration of lower doses in clinical trials to determine if these protective levels of neutralizing antibodies can be achieved in humans; however, protection with a single dose may not be feasible.