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BACKGROUND AND PURPOSE Nox4 is the major NADPH isoform in the kidney and contributes to the pathogenesis of diabetic nephropathy (DN). However, the molecular mechanisms of increased Nox4 expression induced by hyperglycaemia remain to be elucidated. Here, the role of the Connexin32 (Cx32)-Nox4 signalling axis in DN and its related mechanisms were investigated. EXPERIMENTAL APPROACH A diabetic mouse model was induced by low-dose streptozotocin (STZ) combined with a high-fat diet. The effects of Cx32 on Nox4 expression as well as on renal function and fibrosis in STZ-induced diabetic mice were investigated using adenovirus-overexpressing Cx32 and Cx32-deficient mice. The interaction between Cx32 and Nox4 was analysed by co-immunoprecipitation and immunofluorescence assays. KEY RESULTS In this study, we identified that Cx32 was down-regulated in the kidneys of diabetic mice. Overexpression of Cx32 reduced the expression of Nox4 and improved renal function and fibrosis in STZ-induced diabetic mice, whereas Cx32 deficiency had opposite effects. The regulation of Cx32 in fibronectin (FN) expression was not completely dependent on gap junctional intercellular communication involving Cx32. Moreover, Cx32 interacted with Nox4 and reduced the generation of hydrogen peroxide, eventually down-regulating FN expression. Mechanistically, Cx32 decreased Nox4 expression by promoting its K48-linked polyubiquitination. Interestingly, Smurf1overexpression inhibited K48-linked polyubiquitination of Nox4. Furthermore, Cx32 interacted with Smurf1 and inhibited its expression. CONCLUSION AND IMPLICATIONS Cx32 ameliorates renal fibrosis in diabetic mice by promoting K48-linked Nox4 polyubiquitination and degradation via the inhibition of Smurf1 expression. Targeting the Cx32-Nox4 signalling axis may contribute to the development of novel therapeutics for DN.