Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we used an in vitro reconstitution assay to characterize the intra-mitochondrial O- GlcNAc system without potential cytoplasmic confounding effects. We compared the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific O-GlcNAcylation inducer. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 displayed increased O-GlcNAcylation in response to NButGT. This was associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of CI substrates, and a striking reduction of mitochondrial ROS release, which could be related to O- GlcNAcylation of subunits within the NADH dehydrogenase module of CI. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.