Abstract Background: Renin-angiotensin system (RAS) plays a critical role in the progression of renal fibrosis. Angiotensin II type 1 receptor (AT1R) belongs to the B family of G protein coupled receptors (GPCRs) family. β-arrestins are known as negative regula-tors of GPCRs. Recently, β-arrestins have been found to regulate multiple intracellu-lar signaling pathways independent of G proteins. In this study we investigated the role of β-arrestins in regulating extracellular matrix (ECM) synthesis in renal fibrosis. Methods: Rat kidney fibroblast cell line (NRK-49F) was treated with β-arrestin bi-ased agonist SII ([1-sar, 4, 8-ile]-angiotensin II), which does not initiate AT1R-G pro-tein signaling. The cells were transfected with recombinant adenoviruses expressing β-arrestin2 gene or siRNA targeting β-arrestin2. Unilateral ureteral obstruction (UUO) model was used in vivo. Results: The mRNA and protein levels of β-arrestin2, not β-arrestin1, were signifi-cantly up-regulated in the UUO kidney tissues. SII induced the binding of β-arrestin2 with AT1R. SII increased the synthesis of collagen I and fibronectin in NRK-49F, which were abolished when pretreated with candesartan (AT1 receptor blocker). Transfection of siRNA targeting β-arrestin2 decreased the effects of SII on the ECM synthesis. Overexpression of β-arrestin2 enhanced SII-stimulated ECM synthesis. SII induced ERK1/2 phosphorylation in NRK-49F. Transfection of siRNA targeting β-arrestin2 inhibited ERK phosphorylation. Overexpression of β-arrestin2 increased ERK1/2 phosphorylation. Conclusions: Our study firstly showed that AT1R-β-arrestin2 pathway signaling play an important role in renal fibrosis, although it was previously believed that AT1R-G proteins pathway play a major role. Targeting β-arrestin2 may be potential therapeutic agents for renal fibrosis.