Foxp3(+) regulatory T cells (Tregs) play a critical role in maintaining immune self-tolerance. Reduced number and activity of Tregs are usually found in autoimmune and inflammatory diseases, and enhancing the differentiation of Tregs may be a promising therapeutic strategy. Some reports suggested an anti-inflammatory and anti-autoimmune potential for fenofibrate, a hypolipidemic drug used worldwide, whose lipid effects are mediated by the activation of peroxisome proliferator-activated receptor α (PPARα). In the present paper, we found that fenofibrate dose-dependently increased transforming growth factor-β and interleukin-2-induced Treg differentiation in vitro, by 1.96-fold from 0 to 20 μM (12.59 ± 1.34% to 24.69 ± 3.03%, P < 0.05). Other PPARα activators, WY14643 (100 μM), gemfibrozil (50 μM), and bezafibrate (30 μM), could not enhance Treg differentiation. In addition, PPARα could not upregulate the promoter activity of the Treg-specific transcription factor Foxp3. Fenofibrate might exert its function by enhancing Smad3 phosphorylation, a critical signal in Treg differentiation, via Akt suppression. Our work reveals a new PPARα independent anti-inflammatory mechanism of fenofibrate in up-regulating mouse Treg differentiation.