Renal interstitial fibrosis (RIF) is the common pathological process of chronic kidney diseases leading inevitably to renal function deterioration. RIF and its preceding epithelial-mesenchymal transition (EMT) are commonly triggered by an early occurring renal inflammation. However, an effective approach to prevent EMT and RIF is still lacking and of urgent need. Recently, the adenosine A2A receptor (A2AR) emerges as a novel inflammation regulator, therefore manipulation of A2AR may suppress the EMT process and as such protect against RIF. To test this hypothesis we applied a unilateral ureteral obstruction (UUO) model of RIF on A2AR knockout mice and their wild-type littermates, combined with the intervention of a selective A2AR agonist, CGS 21680. On days 3, 7 and 14 post-UUO we evaluated the effects of A2AR manipulation on the molecular pathological progresses of RIF, including the cellular component of interstitial infiltration, expression of profibrotic factors, cellular biomarkers of EMT, and collagen deposition of extracellular matrix. Our data demonstrated that activation of A2AR significantly suppressed the deposition of collagen types I and III, reduced the infiltration of CD4+ T lymphocytes, and attenuated the expression of TGF-β1 and ROCK1, which in turn inhibited and postponed the EMT progress. Conversely, genetic inactivation of A2AR exacerbated the aforementioned pathological processes of UUO-induced RIF. Together, activation of A2AR effectively alleviated EMT and RIF in mice, suggesting A2AR as a potential therapeutic target for the treatment of RIF.