MicroRNAs play important roles in the pathogenesis of diabetic nephropathy (DN). In this study, we found that high glucose upregulated miR-27a expression in cultured glomerular mesangial cells and in the kidney glomeruli of streptozotocin (STZ)-induced diabetic rats. miR-27a knockdown prevented high glucose-induced mesangial cell proliferation and also blocked the upregulation of extracellular matrix (ECM)-associated profibrotic genes. Reduction of cell proliferation and profibrotic gene expression by a miR-27a inhibitor depended upon the expression of peroxisome proliferator-activated receptor γ (PPARγ). Further studies showed that miR-27a negatively regulated PPARγ expression by binding to the 3'-untranslated region of rat PPARγ. An antisense oligonucleotide specific to miR-27a (antagomir-27a) significantly reduced renal miR-27a expression in STZ-induced diabetic rats and significantly increased PPARγ levels. Antagomir-27a also reduced kidney ECM accumulation and proteinuria in STZ-induced diabetic rats. These findings suggest that specific reduction of renal miR-27a decreases renal fibrosis, which may be explained in part by its regulation of PPARγ, and that targeting miR-27a may represent a novel therapeutic approach for DN.