The role of ADP-ribosyl cyclases (ADPR-cyclases) in diabetic nephropathy was investigated. ADPR-cyclases synthesize cyclic ADP-ribose (cADPR), a Ca²⁺-mobilizing second messenger, and are stimulated by G protein-coupled receptors. We have previously reported that ADPR-cyclases can be activated by angiotensin II (Ang II) and showed a specific kidney ADPR-cyclase inhibitor, 4,4'-dihydroxyazobenzene (DHAB), can protect Ang II-mediated mesangial cell growth. In this study we examined the preventive effect of DHAB on glomerular injury in streptozotocin (STZ)-induced diabetic mice. Male mice were randomly assigned to normal control and diabetic groups of comparable age. A diabetic group received 45 µg/kg of DHAB for 6 weeks via daily intraperitonial injection. Several nephropathy parameters were improved in the DHAB treated diabetic group compared to the diabetic group including: urinary albumin (diabetic, 44.6 ± 5.1 vs. treated, 33.9 ± 3.9 µg/day), creatinine clearance (diabetic, 0.72 ± 0.03 vs. treated, 0.83 ± 0.04 ml/min/100g), ratio of kidney to body weight (diabetic, 2.5 ± 0.04 vs. treated, 1.4 ± 0.04), and mesangial matrix expansion (diabetic, 13.9 ± 2.2 vs. treated, 8.5 ± 2.0%). These results indicate that kidney function in STZ-induced diabetes was improved by DHAB administration. Furthermore, DHAB inhibited phosphorylation of Akt and NFAT3 nuclear translocation, as well as ADPR-cyclase activity and cADPR production, which were increased in the kidney of diabetic group. In addition, DHAB treatment decreased fibrosis marker protein expression and glomerular hypertrophy in the diabetic kidney. These findings indicate a crucial role of ADPR-cyclase signaling played in renal pathogenesis of diabetes and provide a therapeutic tool for renal diseases.