Proliferation of interstitial fibroblasts is a hallmark of progressive renal fibrosis commonly resulting in chronic kidney failure. The intermediate-conductance Ca²⁺-activated K⁺ channel (K_Ca3.1) has been proposed to promote mitogenesis in several cell types and contribute to disease states characterized by excessive proliferation. Here, we hypothesized that K_Ca3.1 activity is pivotal for renal fibroblast proliferation and that deficiency or pharmacological blockade of K_Ca3.1 suppresses development of renal fibrosis. We found that mitogenic stimulation up-regulated K_Ca3.1 in murine renal fibroblasts via a MEK-dependent mechanism and that selective blockade of K_Ca3.1 functions potently inhibited fibroblast proliferation by G₀/G₁ arrest. Renal fibrosis induced by unilateral ureteral obstruction (UUO) in mice was paralleled by a robust up-regulation of K_Ca3.1 in affected kidneys. Mice lacking K_Ca3.1 (K_Ca3.1^−/−) showed a significant reduction in fibrotic marker expression, chronic tubulointerstitial damage, collagen deposition and αSMA⁺ cells in kidneys after UUO, whereas functional renal parenchyma was better preserved. Pharmacological treatment with the selective K_Ca3.1 blocker TRAM-34 similarly attenuated progression of UUO-induced renal fibrosis in wild-type mice and rats. In conclusion, our data demonstrate that K_Ca3.1 is involved in renal fibroblast proliferation and fibrogenesis and suggest that K_Ca3.1 may represent a therapeutic target for the treatment of fibrotic kidney disease.