Renal fibrosis is a final common pathway of chronic kidney disease. Sustained activation of fibroblast is considered to play a key role in perpetuating renal fibrosis, but the driving force in the perpetuation stage is only partially understood. To date, some investigations have identified specific overexpression of miR-21 in the progression of kidney fibrosis. Nevertheless, the precise role of miR-21 in fibroblast activation remains largely unknown. In this study, we identified miR-21 was significantly upregulated in activated fibroblasts, and maintained itself at constant high level by employing a miR-21/PDCD4/AP-1 auto-regulatory loop. The persistent up-regulated miR-21 depressed Smad7 expression and eventually enhanced TGF-beta1/Smad pathway to promote fibroblast activation. More importantly, we found miR-21 sequestration with miR-21 antagomir or AP-1 inhibitors attenuated UUO-induced renal fibrosis. miR-21-knockout mice also suffered far less interstitial fibrosis in response to kidney injury. Altogether, these data suggest that miR-21 is a main driving force of fibroblast activation and keeps its high expression level by employing a double negative autoregulatory loop. Targeting this aberrantly activated feedback loop may provide new therapeutic strategy in treating fibrotic kidneys.