Abstract Glycerol plays an important role in the adaptation of fungi to various microenvironments and stressors, including heat shock, anoxic conditions and osmotic stress. Glycerol 3-phosphate dehydrogenase (G3PDH) is able to catalyze dihydroxyacetone phosphate to glycerol 3-phosphate (G3P), which is subsequently dephosphorylated into glycerol. However, current knowledge about the functions of G3PDH homologs in glycerol biosynthesis in Aspergillus fumigatus is limited. Here, we show that the A. fumigatus G3PDH gene, gfdA, is crucial for normal colony growth in glucose media under both normoxic and hypoxic conditions. In addition, failure of the overexpression of the gfdA homolog, gfdB, to rescue the phenotype of a gfdA null mutant suggests that gfdA plays a predominant role in the synthesis of G3P and glycerol. However, in a wild-type background, overexpressing either gfdA or gfdB is able to significantly enhance biomass production of mycelia, suggesting that gfdA and gfdB have similar functions in promoting the use of glucose. Interestingly, overexpression of the gene encoding the predicted glycerol kinase, GlcA, which is capable of phosphorylating glycerol to form G3P, significantly rescues the growth defects of gfdA null mutants in glucose media, indicating that the growth defects of gfdA null mutants might be due to the absence of G3P rather than glycerol. Moreover, Western blotting analysis revealed that gfdA is inducibly expressed by osmotic mediators. However, in the absence of gfdA, osmotic stress can rescue colony growth defects and allow colonies to partially bypass the gfdA requirement in a high osmolarity glycerol pathway-dependent manner. Therefore, the findings of this study elucidate how saprophytic filamentous fungi have developed pathways distinct from those of budding yeasts to adapt to varied carbon sources and survive environmental stresses.