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/6J mice were randomly divided into control, DN and tubastatin A groups. Mice in DN and tubastatin A groups were intraperitoneally injected with 80 mg/ kg STZ daily for 3 days after removal of one kidney. The tubastatin A group received tubastatin A treatments every 3 days for 8 weeks. RNA-sequencing of differentially expressed genes was performed in kidney tissue of DN and tubastatin A groups. Mitochondrial damage was assessed by transmission electron microscopy. ROS levels in kidney tissue were estimated by DHE staining. Mouse glomerular endothelial cells (mGECs) were exposed to high glucose (HG) medium or 40 mmol/ L mannitol (control) with or without tubastatin A treatment. Western blot was used to analyze expression of HDAC6, kidney injury marker KIM1, and Epithelial-Mesenchymal Transition (EMT) markers. Flow cytometry was used to detect mitochondrial ROS and apoptosis in cells. Results HDAC6 expression was upregulated in DN mouse kidney tissue and mGECs exposed to HG, which was consistent with the increased level 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 DMSO as the control, tubastatin A significantly decreased expression of KIM1, HDAC6, α-SMA, N-cadherin and vimentin (P<0. 05) and upregulated E-cadherin expression (P<0. 05) in mGECs under HG treatment. RNA-sequencing revealed enrichment of genes related to ECM-receptor interactions and the tricarboxylic acid cycle in kidney tissue of the Tubastatin A group compared with the findings in the DN group. Transmission electron microscopy showed that the proportion of damaged mitochondria in glomerular endothelial cells in the Tubastatin A group was significantly lower than that in the DN group (P<0. 01). The ROS level in kidney tissue of the Tubastatin A group was lower than that in the DN group (P< 0. 01). In mGECs, tubastatin A treatment downregulated HG-induced mitochondrial ROS levels (P<0. 01) and reduced apoptosis (P<0. 05). Conclusions Tubastatin A ameliorates HG-induced glomerular endothelial cell injury and DN progression, and its mechanism is related to protection of mitochondrial homeostasis and inhibition of EMT.