AbstractIn Aspergillus nidulans, there are two putative glycerol 3‐phosphate dehydrogenases encoded by the genes gfdA and gfdB, while the genome of the osmophilic Aspergillus glaucus harbors only the ortholog of the A. nidulans gfdA gene. Our aim was to insert the gfdB gene into the genome of A. glaucus, and we reached this goal with the adaptation of the Agrobacterium tumefaciens‐mediated transformation method. We tested the growth of the gfdB‐complemented A. glaucus strains on a medium containing 2 mol l‒1 sorbitol in the presence of oxidative stress generating agents such as tert‐butyl hydroperoxide, H2O2, menadione sodium bisulfite, as well as the cell wall integrity stress‐inducing agent Congo Red and the heavy metal stress eliciting CdCl2. The growth of the complemented strains was significantly higher than that of the wild‐type strain on media supplemented with these stress generating agents. The A. nidulans ΔgfdB mutant was also examined under the same conditions and resulted in a considerably lower growth than that of the control strain in all stress exposure experiments. Our results shed light on the fact that the gfdB gene from A. nidulans was also involved in the stress responses of the complemented A. glaucus strains supporting our hypothesis on the antioxidant function of GfdB in the Aspergilli. Nevertheless, the osmotolerant nature of A. glaucus could not be explained by the lack of the gfdB gene in A. glaucus, as we hypothesized earlier.