Sporotrichosis is a subcutaneous mycosis of humans and other mammals, caused by dimorphic species of the genus Sporothrix. In Brazil, human disease is broadly linked to transmission by infected cats and is mainly caused by Sporothrix brasiliensis, Sporothrix schenckii and Sporothrix globosa. In this study, we used a nanoscale liquid chromatography coupled with tandem mass spectrometry approach to provide the yeast proteomic profiles of S. brasiliensis, S. schenckii and S. globosa. From a total of 247 identified proteins, 137 were found as differentially expressed. Functional classification revealed that most are related to carbohydrate and amino acid metabolism as well as stress response. Our data indicate that S. brasiliensis metabolism is distinct of that of S. schenckii and S. globosa, mainly regarding amino acid metabolism and cell wall remodeling, which are induced in the former. Enzymes belonging to glycolytic pathway are, on the other hand, up-regulated in S. schenckii and S. globosa. These findings may explain the previously described more virulent character of S. brasiliensis. Besides complementing genomic comparisons already published, this first comparative proteomic study provided information that indicates new aspects of Sporothrix species metabolism as well as offers information that may be useful in the development of prospective functional studies.