Abstract Pneumocystis(PC) pneumonia remains the most common serious opportunistic infection in patients with HIV/AIDS and is re-emerging in non-HIV infected immunosuppressed patients. Clearance of PC occurs through CD4+ T-cells with antibodies acting as one documented mechanism of clearance, although protective antibody epitopes have yet to be elucidated. To address the role of CD4+ T-cells, we used transcriptomics in mice over the course of PC infection and we observed a strong eosinophil signature in CD4-replete animals by RNA sequencing and by BAL analysis. Hypereosinophilic CD4-depleted mice receiving pIL5 via hydrodynamic injection had increased eosinophils in the lung and decreased PC burden. Finally, mice deficient in eosinophilopoiesis, had no change in burden with pIL5 treatment following CD4-depletion, further implicating eosinophils as novel mediators of immunity against PC. To identify protective antibody epitopes as potential vaccine candidates, we used a life-cycle based approach and sequenced RNA from the ascus and troph life forms of PC and found GSC-1 (ascus) and Meu10 (troph) were differentially regulated. Both GSC-1 and Meu10 generate strong humoral immune responses. Meu10 peptide vaccination decreases PC burden in CD4+ T-cell depleted animals, while immunization with recombinant GSC-1 failed to provide complete protection in a challenge model. Future studies will evaluate a multivalent life-cycle based vaccine in both a PC challenge model and a co-housing model.