Defective mitochondrial proteins are emerging as major contributors to human disease. Nicotinamide nucleotide transhydrogenase (NNT), a widely expressed mitochondrial protein, has a crucial role in the defence against oxidative stress. NNT variations have recently been reported in patients with familial glucocorticoid deficiency (FGD) and in patients with heart failure. Moreover, knockout animal models suggest that NNT has a major role in diabetes mellitus and obesity. In this study, we used experimental structures of bacterial transhydrogenases to generate a structural model of human NNT (H-NNT). Structure-based analysis allowed the identification of H-NNT residues forming the NAD binding site, the proton canal and the large interaction site on the H-NNT dimer. In addition, we were able to identify key motifs that allow conformational changes adopted by domain III in relation to its functional status, such as the flexible linker between domains II and III and the salt bridge formed by H-NNT Arg882 and Asp830. Moreover, integration of sequence and structure data allowed us to study the structural and functional effect of deleterious amino acid substitutions causing FGD and left ventricular non-compaction cardiomyopathy. In conclusion, interpretation of the function–structure relationship of H-NNT contributes to our understanding of mitochondrial disorders.