Autophagy is a cell self-renewal process that relies on the degradation of the cytoplasmic proteins or organelles of lysosomes and is associated with development of numerous diseases. However, the therapeutic effect of autophagy inhibition on hyperuricemic nephropathy (HN) and the underlying mechanisms are still unknown. Here, we investigated the effect of delayed treatment with 3-methyladenine (3-MA), a specific autophagy inhibitor, on the development of HN in a rat model. Administration of 3-MA at 21 days following after uric acid injury protected kidney from hyperuricemic-related injuries, as demonstrated by improving renal dysfunction and architecture damage, blocking Beclin-1 and LC3II/I and decreasing the number of autophagic vacuoles. Late treatment with 3-MA was also effective in attenuating renal fibrosis as evidenced by reducing ECM protein deposition, blocking epithelial-to-mesenchymal transition (EMT) and decreasing the number of renal epithelial cells arrested at the G2/M phase of cell cycle. Injury to the kidney resulted in increased expression of TGFβ receptor I, and phosphorylation of Smad3, 3-MA significantly abrogated all these responses. Moreover, inhibition of autophagy suppressed mitochondrial fission, downregulated the expression of Dynamin-related protein 1 (Drp-1), Cofilin and F-actin, and alleviated cell apoptosis. Finally, 3-MA effectively blocked STAT3 and NF-κB phosphorylation and suppressed infiltration of macrophages and lymphocytes as well as release of multiple profibrogenic cytokines/chemokines in the injured kidney. Taken together, these findings indicate that hyperuricemia-induced autophagy is critically involved in the activation of renal fibroblasts, EMT, mitochondrial fission and apoptosis of tubular epithelial cells and development of renal fibrosis. Thus, this study provides evidence for autophagy inhibitors as the treatment of HN patients.