Updated project metadata. Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America. Few virulence factors have been identified for this pathogen. In this study, we quantitatively evaluated the protein composition of P. brasiliensis in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, we identified and quantified 317 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 214 identified proteins were over-expressed in the virulent fungus. Alcohol dehydrogenase, mitochondrial peroxiredoxin PRX1 and protein vacuolar ATP synthase catalytic subunit A were up-regulated in virulent Pb18 in both media, suggesting potential roles of these proteins in virulence regulation in P. brasiliensis. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and to understand how Paracoccidioides modulates the host-pathogen interaction to its advantage. Data are available via ProteomeXchange Consortium with identifier PXD000804.