Dry eye and diabetic keratopathy represent the major diabetic complications in ocular surface. Here we found that diabetic mice exhibited the early onset of reduced tear secretion and lacrimal gland weight compared to the symptoms of diabetic keratopathy. Considering to the high bioenergetic needs in lacrimal gland and cornea, we hypothesized that hyperglycemia may cause different severity of mitochondrial bioenergetic deficit between them. Through the measurement of oxygen consumption rate (OCR) and basal extracellular acidification rate (ECAR), we found the apparent alterations of mitochondrial bioenergetic profiles in diabetic lacrimal gland and cornea, accompanied with the mtDNA damage and copy number reduction, as well as the reduced glutathione content. Comparative analysis revealed that mouse lacrimal gland cells exhibited 2-3 folds higher of basal, ATP production, maximal OCR and basal ECAR than corneal epithelial cells in normoglycemia. However, the differences were slightly significant or even not detected in hyperglycemia. Accordingly, the mitochondrial bioenergetic metabolism of lacrimal gland was more compromised than that of corneal epithelium in diabetic mice. Through the administration of mitochondrial-targeted antioxidant SkQ1, the severity of dry eye and diabetic keratopathy was significantly attenuated with the improved mitochondrial function. These results indicate that the susceptibility of mitochondrial bioenergetic deficit in diabetic lacrimal gland may contribute to the early onset of dry eye, while mitochondria-targeted antioxidant possesses therapeutic potential for diabetic dry eye and keratopathy.