Podocyte dysfunction is important in the onset and development of diabetic nephropathy (DN). Histone deacetylases (HDACs) have been recently proved to play critical roles in the pathogenesis of DN. As one subtype of the class IIa HDACs, HDAC9 is capable to repress/de-repress their target genes in tumor, inflammation, atherosclerosis and metabolic diseases. In the present study, we investigate whether HDAC9 is involved in the pathophysiologic process of DN, especially the podocyte injury. Firstly, we explored the expression patterns and localization of HDAC9 and found that HDAC9 expression was significantly up-regulated in high glucose (HG)-treated mouse podocytes, as well as kidney tissues from diabetic db/db mice and patients with DN. Secondly, knockdown of HDAC9 in mouse podocytes significantly suppressed HG-induced reactive oxygen species (ROS) generation, cell apoptosis and inflammation through JAK2/STAT3 pathway and reduced the podocytes injury by decreasing the expression levels of Nephrin and Podocin. Moreover, in diabetic db/db mice, silencing of HDAC9 attenuated the glomerulosclerosis, inflammatory cytokine release, podocyte apoptosis and renal injury. Collectively, these data indicate that HDAC9 may be involved in the process of DN, especially podocyte injury. Our study suggest that inhibition of HDAC9 may have a therapeutic potential in DN treatment.