Cryptococcus neoformans is a fungal pathogen most commonly causing meningitis in immunocompromised patients. Current therapies are inadequate, and novel antifungal targets are needed. We have identified by proteomics two thiol peroxidases that are differentially expressed at 37°C, the temperature of the mammalian host. Consistent with their antioxidant role, we show that the genes encoding these thiol-specific antioxidants, TSA1 and TSA3, are transcriptionally induced when C. neoformans is exposed to hydrogen peroxide. Genome sequence analysis of C. neoformans revealed a third thiol peroxidase, TSA4. We constructed single, double and triple mutants of the thiol peroxidase genes through homologous recombination and analysed their function by comparing the growth of these mutants with that of the wild-type strain. The tsa1Δ mutant shows sensitivity to hydrogen peroxide and t-butylhydroperoxide, as well as significant growth retardation at 25°C and 38.5°C. The tsa1Δ mutant is also sensitive to NO, demonstrating a link between oxidative and nitrosative stress pathways. In two mouse models of cryptococcosis, the tsa1Δ mutant is significantly less virulent.