The oxidation of free and protein-bound methionine into methionine sulfoxide is a frequently occurring modification caused by ROS. Most organisms express methionine sulfoxide reductases (MSR enzymes) to repair this potentially damaging modification. Humans express three different MSRB enzymes which reside in different cellular compartments. In this study, we have explored the specificity of the human MSRB enzymes both by in silico modeling and by experiments on oxidized peptides. We found that MSRB1 is the least specific MSRB enzyme, which is in agreement with the observation that MSRB1 is the only MSRB enzyme found in the cytosol and the nucleus, and therefore requires a broad specificity to reduce all possible substrates. MSRB2 and MSRB3, which are both found in mitochondria, are more specific but because of their co-occurrence they can likely repair all possible substrates.