Cryptococcus neoformans is an encapsulated yeast that causes a life-threatening meningoencephalitis in immunocompromised individuals. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this pathogen. This trait is controlled in part by the Ca²(+)-calcineurin pathway, which senses and utilizes cytosolic calcium for signaling. In the present study, the identification of the C. neoformans gene VCX1, which encodes a vacuolar calcium exchanger, is reported. The VCX1 knockout results in hypersensitivity to the calcineurin inhibitor cyclosporine A at 35°C, but not at 30°C. Furthermore, high concentrations of CaCl₂ lead to growth inhibition of the vcx1 mutant strain only in the presence of cyclosporine A, indicating that Vcx1 acts in parallel with calcineurin. The loss of VCX1 does not influence cell wall integrity or capsule size but decreases secretion of the major capsular polysaccharide glucuronoxylomannan (GXM) in culture supernatants.Vcx1 also influences C. neoformans phagocytosis by murine macrophages and is required for full virulence in mice. Analysis of cellular distribution by confocal microscopy confirmed the vacuolar localization of Vcx1 in C. neoformans cells.