In the present study, we examined the characteristics of cDNA, the regulation of the gene expression of Paracoccidioides brasiliensis MLS (Pbmls), and the enzymatic activity of the protein P. brasiliensis MLS (PbMLS) from the P. brasiliensis Pb01 isolate. Pbmls cDNA contains 1617 bp, encoding a protein of 539 amino acids with a predicted molecular mass of 60 kDa. The protein presents the MLSs family signature, the catalytic residues essential for enzymatic activity and the peroxisomal/glyoxysomal targeting signal PTS1. The high level of Pbmls transcript observed in the presence of two-carbon (2C) sources suggests that in P. brasiliensis, the primary regulation of carbon flux into the glyoxylate cycle (GC) was at the level of the Pbmls transcript. The gene expression, protein level, and enzymatic activity of Pbmls were highly induced by oxalurate in the presence of glucose and by proline in the presence of acetate. In the presence of glucose, the gene expression, protein level, and enzymatic activity of Pbmls were mildly stimulated by proline. Our results suggested that PbMLS condenses acetyl-CoA from both 2C sources (GC) and nitrogen sources (from proline and purine metabolism) to produce malate. The regulation of Pbmls by carbon and nitrogen sources was reinforced by the presence of regulatory motifs CREA and UIS found in the promoter region of the gene.