objective: To investigate the protective effect and mechanism of histone deacetylase 6 (HDAC6) specific small molecule inhibitor Tubastatin A on renal injury in diabetic nephropathy (DN) mice. Methods: C57BL/6 mice were randomly divided into 3 groups: control group, DN group and Tubastatin A group. Mice in the DN group and Tubastatin A group were intraperitoneally injected with燬TZ?80爉g/kg) daily for 3 days after the removal of one kidney. Tubastatin A group received Tubastatin A treatment every 3 days for 8 weeks. RNA sequencing analysis of differentially expressed genes in kidney tissue of DN group and Tubastatin A group. Mitochondrial damage was assessed by transmission electron microscopy, and ROS levels in kidney tissue were estimated by DHE staining. Mouse glomerular endothelial cells (mGEC) were exposed to high glucose medium (HG) or 40 mM mannitol (control) with or without Tubastatin A treatment. Western blot analysis was used to analyze the expression of HDAC6, kidney injury markers KIM1 and EMT markers, and flow cytometry was used to detect mitochondrial ROS and apoptosis in cells. Results: HDAC6 expression was up-regulated in DN mouse kidney tissue and mGEC cells exposed to HG, consistent with increased levels of KIM1. Histological analysis showed significant morphological changes in DN mice, including glomerular hypertrophy, mesangial matrix accumulation, glomerular basement membrane thickening, tubular basement membrane thickening and the presence of glomerular, intertubular fibrosis; Tubastatin A treatment alleviated these changes. Compared with control DMSO, Tubastatin A significantly decreased the expression of KIM1, HDAC6, α-SMA, N-cadherin, vimentin (P<0.05) and up-regulated the expression of E-cadherin (P<0.05) in mGEC cells under HG treatment. RNA-sequencing revealed the enrichment of genes related to ECM-receptor interaction and tricarboxylic acid (TCA) cycle in the kidney tissue of Tubastatin A mice compared with DN mice. Transmission electron microscopy showed that the proportion of damaged mitochondria in glomerular endothelial cells in Tubastatin A group was significantly lower than that in DN group (P<0.01). DHE staining showed that the level of ROS in the kidney tissue of Tubastatin A group was lower than that of DN group (P<0.01). In mGEC cells, Tubastatin A treatment down-regulated HG-induced mitochondrial ROS levels in mGEC cells (P<0.01), and reduced apoptosis (P<0.05). Conclusion: Tubastatin A ameliorates HG-induced glomerular endothelial cell injury and DN progression, and its mechanism is related to the protection of mitochondrial homeostasis and inhibition of EMT.