The kidney is the second most mitochondria-rich organ, and effective energy production is essential for maintaining a healthy kidney function. Dysfunctional mitochondrial bioenergetics such as defective respiratory chain function, and compromised mitochondrial morphology are thought to be central to the development of diabetic kidney disease (DKD). However, the role of systemic glucose concentrations in the development of this disease remains unknown. The effects of different blood glucose concentrations in the kidney of a rodent model of streptozotocin-induced diabetes, including respiratory chain function, mitochondrial dynamics and the mitoproteomic landscape were investigated. Diabetic rats were treated with either an intensive or a conventional insulin therapy for 8 weeks, resulting in blood glucose levels of ~20mmol/l (moderate hyperglycaemia, MHG) or ~30mmol/l (severe hyperglycaemia, SHG), respectively, and were compared to controls. Albuminuria and glomerulosclerosis, representing hallmarks of diabetic kidney disease, were induced in both MHG and SHG. However, intensive insulin therapy (MHG) afforded renoprotection, leading to improved glomerular hyperfiltration, and decreased albuminuria, glomerular injury, and renal fibrosis, compared to rats with SHG. Kidney mitochondrial bioenergetics were altered in both groups, and a decline in complex I activity, increased citrate synthase activity and reactive oxygen species generation, and greater mitochondrial fragmentation within the proximal tubular epithelial cells, were reported. Lowering blood glucose to MHG improved the mitochondrial phenotype. Quantitative mitochondrial proteomics revealed a clear change in the mitochondrial signature induced by hyperglycaemia. Distinct differential landscapes were observed in SHG rats compared with control and MHG; gene ontology analysis showed upregulation of ketone, fatty acid, and glutathione metabolic processes, and downregulation of transmembrane transport and protein translation pathways. These data demonstrate that hyperglycemia induces alterations in mitochondrial bioenergetics and the mitoproteomic landscape, and correlate with the severity of renal lesion, suggesting that severe hyperglycaemia is a key determinant of mitochondrial homeostasis in DKD.