Activation of (pro)renin receptor (PRR) is involved in the progression of chronic kidney disease. However, the role of indoxyl sulfate, a uremic toxin, in the activation of PRR is not clear. The present study aimed to clarify the role of indoxyl sulfate in activation of PRR, in relation to renal expression of fibrotic genes. Renal expression of PRR and renin/prorenin was up-regulated in chronic kidney disease rats compared with normal rats, whereas AST-120 suppressed these expression by reducing serum levels of indoxyl sulfate. Furthermore, administration of indoxyl sulfate to normotensive and hypertensive rats increased renal expression of PRR and renin/prorenin. Indoxyl sulfate induced expression of PRR and prorenin in cultured human proximal tubular cells (HK-2 cells). Indoxyl sulfate-induced PRR expression was inhibited by small interfering RNAs of signal transducer and activator of transcription 3 (Stat3) and nuclear factor-κB p65 in proximal tubular cells. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed indoxyl sulfate-induced PRR expression in proximal tubular cells. N-acetylcysteine prevented indoxyl sulfate-induced phosphorylation of Stat3 in proximal tubular cells. PRR small interfering RNA inhibited indoxyl sulfate-induced expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Taken together, indoxyl sulfate-induced up-regulation of prorenin expression and activation of PRR through production of reactive oxygen species and activation of Stat3 and nuclear factor-κB play an important role in the expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Thus, indoxyl sulfate-induced activation of prorenin/PRR might be involved in renal fibrosis.