Methionine sulfoxide reductases (Msr) are repair proteins, which provide an important part of the cellular antioxidant defense. Methionine oxidation results in a diasteromeric mixture of methionine-S-sulfoxide (Met-S-O) and methionine-R-sulfoxide (Met-R-O), requiring MsrA enzymes to reduce Met-S-O and MsrBs to reduce Met-R-O. Here we explored the filamentous fungus Aspergillus nidulans as a model organism for studies on Msr enzymes. The Msr system of A. nidulans was found to comprise three enzymes: AnMsrA, AnMsrB, and AnfRMsr. Single-knockout strains for all three enzymes showed reduced viability under oxidative stress conditions (H2O2, Chloramine-T, menadione). In addition, all three msr genes were transcriptionally upregulated by oxidative stress, underscoring the relevance of the Msr enzymes for the stress response of the fungus. Biochemical characterization of recombinant AnMsrs showed that MsrA reduces both, free and peptide-bound Met-S-O, while MsrB reduces Met-R-O with a strong preference for peptide substrates over free Met-R-O. By contrast, fRMsr is an MsrB-type enzyme that only accepts free Met-R-O as substrate. The predicted active centers for all three enzymes were confirmed by site-directed mutagenesis of the catalytic Cys residues. The peptide substrate specificity of MsrA and MsrB from A. nidulans and of the corresponding human Msrs was studied, using a set of peptides with systematic variation of the residues flanking the oxidized methionine. MsrA activity was strongly reduced by acidic residues in these neighboring positions whereas MsrBs were less sensitive to the type of flanking amino acids. Site-directed mutagenesis around the catalytic cysteine of AnMsrA identified two negatively charged residues (Glu99 and Asp134 in) to have strong impact on the MsrA peptide selectivity. A. nidulans proved as a suitable eukaryotic model organism for studies of Msr enzymes that allows easy genetic manipulation and functional analysis.