Podocyte injury plays a critical role in the progression of diabetic kidney disease (DKD), but the underlying cellular and molecular mechanisms remain poorly understanding. MicroRNAs (miRNAs) can disrupt gene expression by inducing translation inhibition and mRNA degradation, and recent evidence has shown that miRNAs may play a key role in many kidney diseases. In this study, we identified miR-4645-3p by global transcriptome expression profiling as one of the major downregulated miRNAs in high glucose-cultured podocytes. Moreover, whether DKD patients or STZ-induced diabetic mice, expression of miR-4645-3p was also significantly decreased in kidney. In the podocytes cultured by normal glucose, inhibition of miR-4645-3p expression promoted mitochondrial damage and podocyte apoptosis. In the podocytes cultured by high glucose (30 mM glucose), overexpression of miR-4645-3p significantly attenuated mitochondrial dysfunction and podocyte apoptosis induced by high glucose. Furthermore, we found that miR-4645-3p exerted protective roles by targeting Cdk5 inhibition. In vitro, miR-4645-3p obviously antagonized podocyte injury by inhibiting overexpression of Cdk5. In vivo of diabetic mice, podocyte injury, proteinuria, and impaired renal function were all effectively ameliorated by treatment with exogenous miR-4645-3p. Collectively, these findings demonstrate that miR-4645-3p can attenuate podocyte injury and mitochondrial dysfunction in DKD by targeting Cdk5. Sustaining the expression of miR-4645-3p in podocytes may be a novel strategy to treat DKD.