Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a facultative intracellular human pathogen that can persist within macrophage phagolysosomes, indicating that the fungus has evolved defense mechanisms in order to survive under nutritionally poor environments. The analysis of P. brasiliensis transcriptome revealed several virulence factor orthologs of other microorganisms, including the glyoxylate cycle genes. This cycle allows the utilization of two-carbon (C2) compounds as carbon source in gluconeogenesis. Semiquantitative RT-PCR analyses revealed that these genes were upregulated when P. brasiliensis was recovered from murine macrophages, without any additional in vitro growth. The induction of this cycle, in response to macrophage microenvironments, was shown to be coordinated with the upregulation of the gluconeogenic phosphoenolpyruvate carboxykinase gene. In addition, assays employing RNA extracted from P. brasiliensis grown in a medium with acetate instead of glucose also showed increased levels of glyoxylate cycle transcripts. Our main results suggest that P. brasiliensis uses the glyoxylate cycle as an important adaptive metabolic pathway.