The safety, immunogenicity and efficacy of recombinant Ag2/PRA106 + CSA chimeric fusion protein (CFP) vaccine in ISS/Montanide adjuvant-administered intramuscular (IM) was assessed in adult female cynomolgus macaques challenged with Coccidioides posadasii. Animals received three immunizations with either 5 μg CFP, 50-μg CFP, or adjuvant alone and were challenged 4 weeks following the final immunization. Although significant antibody response was produced in response to vaccination, there were no discernable adverse effects, suggesting that the vaccine was well tolerated. Upon intratracheal challenge, all animals showed evidence of disease. Two animals that received 5-μg doses of CFP were euthanatized prior to the study's end because of severe symptoms. Animals vaccinated with 50-μg doses of CFP showed evidence of enhanced sensitization compared to adjuvant controls and animals vaccinated with 5-μg doses of CFP. This was based on higher serum anti-CFP titers, enhanced secretion of interferon-gamma (IFN-γ) from stimulated bronchoalveolar lavage mononuclear cells (BALMC), reduced pulmonary radiologic findings following intratracheal challenge, reduced terminal complement fixation titers, and reduced necropsy findings. Overall the vaccine was well tolerated, induced sensitization, and resulted in a protective response when given at the higher 50-μg dose. Additional experiments may be needed to optimize the vaccination and to confer greater protection against lethal challenge.