O-GlcNAcylation, a ubiquitous post-translational modification, rapidly modulates protein activity through the reversible addition and removal of O-GlcNAc groups from serine or threonine residues in target proteins, and is involved in multiple metabolic pathways. With the discovery of enzymes and substrates for O-GlcNAc cycling in mitochondria, mitochondrial O-GlcNAc modification and its regulatory role in mitochondrial function deserve extensive attention. Adaptive regulation of the O-GlcNAc cycling in response to energy perturbations is demonstrated to be important in maintaining mitochondrial homeostasis. Dysregulation of O-GlcNAcylation in mitochondria has been associated with various mitochondrial dysfunctions, such as abnormal mitochondrial dynamics, reduced mitochondrial biosynthesis, disruption of the electron transport chain, oxidative stress and the calcium paradox, as well as activation of mitochondrial apoptosis pathways. Here, we outline the current understanding of O-GlcNAc modification in mitochondria and the key discovery of O-GlcNAcylation in regulating mitochondrial network homeostasis. This review will provide insights into targeting mitochondrial O-GlcNAcylation, as well as the mechanisms linking mitochondrial dysfunction and disease.