Diabetic nephropathy (DN) is the most common cause of end-stage renal disease. Although numerous reports have demonstrated a correlation between epithelial-mesenchymal transition (EMT) and renal fibrosis, how these processes lead to tubular dysfunction remains unclear. Here, we show that FOXO3a protects kidneys from injury in type II DN by increasing Sirt6 expression, which deacetylates Smad3 and inhibits its transcriptional activity. The results showed that progressive EMT in the kidneys from db/db mice is associated with Sirt6 downregulation and involved in tubular injury and dysfunction. The reduction of Sirt6 levels in db/db mice resulted in progressive kidney injury, indicating the protective role of Sirt6. Furthermore, Sirt6 was shown to directly bind to Smad3, a key downstream mediator of TGF-, and could deacetylate it to inhibit its nuclear accumulation and transcriptional activity in HK2 cells. Besides, we demonstrate that FOXO3a activates Sirt6 expression by binding to its promoter. shRNA-induced FOXO3a knockdown in the kidneys of db/db mice exacerbated tubular injury and renal function loss. Mechanistically, FOXO3a protects against kidney injury in type II DN through the Sirt6/Smad3 axis. Thus, the pharmacological targeting of FOXO3a-mediated Sirt6/Smad3 signaling pathways may provide a novel strategy for treating type II DN.