Renal fibrosis is the final common pathway leading to decreased renal function. No therapy has been established to prevent it. In order to establish a therapeutic approach and target molecule for renal fibrosis, we investigated the effects of Smad4 knockdown by siRNAs on renal fibrosis in vivo. Renal fibrosis mice were produced by single intraperitoneal injection of folic acid. siRNAs targeted to Smad4 (Smad4-siRNAs) (5 nmol) were injected into each mouse by systemic tail vein injection three times per week. Non-targeted siRNAs (control-siRNAs) were injected in the same way for a control group. The siRNAs were delivered to the interstitial fibrous area and tubules. Smad4-siRNAs significantly knocked down Smad4 expression and inhibited renal fibrosis. They also inhibited α-SMA-positive myofibroblasts. Control-siRNAs did not show these effects. The results of this study suggest that Smad4 knockdown is one of the crucial therapeutic options for the prevention of renal fibrosis in vivo.