The glyoxylate cycle plays an essential role for anaplerosis of oxaloacetate during growth of microorganisms on carbon sources such as acetate or fatty acids and has been shown to contribute to virulence of several pathogens. Here, we investigated the transcriptional and post-translational regulation of the glyoxylate cycle key enzyme isocitrate lyase (PbICL) in the human pathogenic fungus Paracoccidioides brasiliensis. Although sequence analyses on fungal isocitrate lyases revealed a high phylogenetic conservation, their regulation seems to differ significantly. Closely related Aspergillus species regulate the glyoxylate cycle at the transcriptional level, whereas Pbicl was constitutively expressed in yeast cells. However, only low PbICL activity was detected when cells were grown in the presence of glucose. Two-dimensional gel analyses with subsequent antibody hybridization revealed constitutive production of PbICL, but low PbICL activity on glucose coincided with extensive protein phosphorylation. Since an in vitro dephosphorylation of PbICL from glucose grown cells strongly increased ICL activity and resembled the phosphorylation pattern of highly active acetate grown cells, post-translational modification seems the main mechanism regulating PbICL activity in yeast cells. In agreement, a transfer of yeast cells from glucose to acetate medium increased PbICL activity without requirement of de novo protein synthesis. Thus, inactivation of PbICL by phosphorylation is reversible, denoting a new strategy for the rapid adaptation to changing environmental conditions.