To determine whether TLR9 signaling contributes to the development of the adaptive immune response to cryptococcal infection, wild-type (TLR9+/+) and TLR9 knockout (TLR9-/-) BALB/c mice were infected intratracheally with 10(4) C. neoformans 52D. We evaluated 1) organ microbial burdens, 2) pulmonary leukocyte recruitment, 3) pulmonary and systemic cytokine induction, and 4) macrophage activation profiles. TLR9 deletion did not affect pulmonary growth during the innate phase, but profoundly impaired pulmonary clearance during the adaptive phase of the immune response (a 1000-fold difference at week 6). The impaired clearance in TLR9-/- mice was associated with: 1) significantly reduced CD4(+), CD8+ T cell, and CD19+ B cell recruitment into the lungs; 2) defects in Th polarization indicated by altered cytokine responses in the lungs, lymphonodes, and spleen; and 3) diminished macrophage accumulation and altered activation profile, including robust up-regulation of Arg1 and FIZZ1 (indicators of alternative activation) and diminished induction of inducible nitric oxide synthase (an indicator of classical activation). Histological analysis revealed defects in granuloma formation and increased numbers of intracellular yeast residing within macrophages in the lungs of TLR9-/- mice. We conclude that TLR9 signaling plays an important role in the development of robust protective immunity, proper recruitment and function of effector cells (lymphocytes and macrophages), and, ultimately, effective cryptococcal clearance from the infected lungs.