Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. Here, we evaluated the role of PFD in regulation of the extracellular matrix. In mouse mesangial cells, PFD decreased TGF-beta promoter activity, reduced TGF-beta protein secretion, and inhibited TGF-beta-induced Smad2-phosphorylation, 3TP-lux promoter activity, and generation of reactive oxygen species. To explore the therapeutic potential of PFD, we administered PFD to 17-wk-old db/db mice for 4 wk. PFD treatment significantly reduced mesangial matrix expansion and expression of renal matrix genes but did not affect albuminuria. Using liquid chromatography with subsequent electrospray ionization tandem mass spectrometry, we identified 21 proteins unique to PFD-treated diabetic kidneys. Analysis of gene ontology and protein-protein interactions of these proteins suggested that PFD may regulate RNA processing. Immunoblotting demonstrated that PFD promotes dosage-dependent dephosphorylation of eukaryotic initiation factor, potentially inhibiting translation of mRNA. In conclusion, PFD is renoprotective in diabetic kidney disease and may exert its antifibrotic effects, in part, via inhibiting RNA processing.