Abnormal Wnt5a expression, mitochondrial abnormalities and calcium overload have been detected in many metabolic diseases. However, the association of Wnt5a-Ca2+ and mitochondrial dysfunction in diabetic nephropathy (DN) progression remains unknown. We used streptozotocin-induced DBA2/J male mice as a DN model. The mice were treated with losartan (10 mg/kg/d*12 w) or losartan (10 mg/kg/d*12 w) + levamlodipine (5 mg/kg/d*12 w). High glucose (HG) (40 mmol/L)-induced HK-2 cells were used for in vitro experiments. Wnt5a and mitochondrial calcium uniporter (MCU) expression, mitochondrial dynamics, morphological changes and Ca2+ concentration were detected in different groups. Levamlodipine, a kind of calcium channel blocker, in combination with losartan ameliorated tubular injury and reversed mitochondrial fragmentation and dynamic dysfunction more efficiently than losartan alone in diabetic mice. Wnt5a induced Ca2+ uptake and aggravated mitochondrial fusion-fission disorder in HG-stimulated HK-2 cells. In addition, increased MCU formation was found in the mitochondria of tubular cells under HG stimulation and was upregulated by the activation of the Wnt5a-Ca2+ pathway. Our study showed that the Wnt5a-Ca2+ signalling pathway was involved in Ca2+ overload-induced mitochondrial dysfunction possibly through MCU in tubular injury and DN progression. A calcium channel blocker in combination with a renin-angiotensin system inhibitor (RASi) could be a promising therapeutic strategy in DN patients.