The contribution of microRNA (miRNA) to the pathogenesis of renal fibrosis is not well understood. Here, we investigated whether miRNA modulates the fibrotic process in Munich Wistar Fromter (MWF) rats, which develop spontaneous progressive nephropathy. We analyzed the expression profile of miRNA in microdissected glomeruli and found that miR-324-3p was the most upregulated. In situ hybridization localized miR-324-3p to glomerular podocytes, parietal cells of Bowman's capsule, and most abundantly, cortical tubules. A predicted target of miR-324-3p is prolyl endopeptidase (Prep), a serine peptidase involved in the metabolism of angiotensins and the synthesis of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). In cultured tubular cells, transient transfection with a miR-324-3p mimic reduced Prep protein and activity, validating Prep as a target of this miRNA. In MWF rats, upregulation of miR-324-3p associated with markedly reduced expression of Prep in both glomeruli and tubules, low urine Ac-SDKP, and increased deposition of collagen. ACE inhibition downregulated glomerular and tubular miR-324-3p, promoted renal Prep expression, increased plasma and urine Ac-SDKP, and attenuated renal fibrosis. In summary, these results suggest that dysregulation of the miR-324-3p/Prep pathway contributes to the development of fibrosis in progressive nephropathy. The renoprotective effects of ACE inhibitors may result, in part, from modulation of this pathway, suggesting that it may hold other potential therapeutic targets.