There are at present no published studies providing a global overview of changes in bladder metabolism resulting from diabetes. Such studies have the potential to provide mechanistic insight into the development of diabetic bladder disorder (DBD). In the present study we compared the metabolome of detrusor and urothelial layer in a one month streptozotocin-induced rat model of Type I diabetes with non-diabetic controls. Our studies revealed diabetes caused both common and differential changes in the detrusor and urothelial layer's metabolome. Diabetes resulted in similar changes in levels of previously described diabetic markers in both tissues, such as glucose, lactate, 2-hydroxybutyrate, branched-chain amino acid degradation products, bile acids and 1,5-anhydroglucitol, as well as markers of oxidative stress. In detrusor (but not urothelial layer) diabetes caused activation of the pentose-phosphate and polyol pathways, concomitant with a reduction in the TCA cycle and β-oxidation. Changes in detrusor energy generating pathways resulted in an accumulation of sorbitol which, through generation of advanced glycation end products, is likely to play a central role in the development of DBD. In the diabetic urothelial layer there was decreased flux of glucose via glycolysis and changes in lipid metabolism, particularly prostaglandin synthesis, which also potentially contributes to detrusor dysfunction.