Experimental pulmonary Cryptococcus neoformans infection in BALB/c mice is associated with polarized Th2-type cytokine production, alternative macrophage activation, and severe bronchopneumonia. In contrast, pulmonary infection with a C. neoformans strain that secretes IFN-γ, H99γ, elicits Th1-type cytokine production and classical macrophage activation. Additionally, mice infected with H99γ resolve the acute infection and are subsequently protected against challenge with wild type C. neoformans. The present study characterizes macrophage activation during the protective response to wild type C. neoformans in mice previously immunized with H99γ. We observed increased pulmonary Th1-type cytokine production in lung homogenates and classical macrophage activation as evidenced by enhanced expression of inducible nitric oxide synthase (iNOS) in the lungs of H99γ immunized mice compared to mice given a non-protective immunization with heat-killed C. neoformans (HKCn). Furthermore, macrophages from day 7 post-challenge H99γ immunized mice cultured in vitro were fungistatic against C. neoformans, whereas cryptococcal growth was uncontrolled within macrophages from HKCn immunized mice. Th2-type cytokine production and induction of alternatively activated macrophages were also observed in lungs of HKCn immunized mice during re-challenge. Gene expression arrays showed that classical macrophage activation during challenge infection in H99γ immunized mice was associated with induction of the transcription factor STAT1 and its downstream targets IRF-1, SOCS-1, CXCL9 and CXCL10. These studies demonstrate that protective responses to C. neoformans challenge in immunized mice include classical macrophage activation and enhanced macrophage fungistasis of C. neoformans yeasts. Finally, the classical activation phenotype of protective anti-cryptococcal macrophages is likely mediated via STAT1 signal transduction pathways.