We previously demonstrated that several clinically utilized volatile anesthetics including sevoflurane protected against renal ischemia-reperfusion (IR) injury by reducing necrosis and inflammation in vivo. We also demonstrated that volatile anesthetics produced direct anti-necrotic and anti-inflammatory effects in cultured renal tubules via mechanisms involving the externalization of phosphatidylserine and subsequent release of transforming growth factor (TGF)-β1. In this study, we tested the hypothesis that volatile anesthetic-mediated renal protection requires TGF-β1 and SMAD3 signaling in vivo. We subjected TGF-β1+/+, TGF-β1+/−, SMAD3+/+, or SMAD3−/− mice to renal IR under anesthesia with pentobarbital sodium or with sevoflurane. Although TGF-β1+/+ and SMAD3+/+ mice were significantly protected against renal IR injury under sevoflurane anesthesia with reduced necrosis and inflammation, TGF-β1+/− mice and SMAD3−/− mice were not protected against renal IR with sevoflurane. Furthermore, a neutralizing TGF-β1 antibody blocked renal protection with sevoflurane in TGF-β1+/+ mice. Sevoflurane caused nuclear translocation of SMAD3 and reduced the TNF-α-induced nuclear translocation of NF-κB in primary cultures of proximal tubules from TGF-β1+/+ but not in TGF-β1+/− mice. Finally, sevoflurane protected against necrosis induced with hydrogen peroxide in primary cultures of proximal tubules from TGF-β1+/+ mice or SMAD3+/+ mice but not in proximal tubules from TGF-β1+/− or SMAD3−/− mice. Therefore, we demonstrate in this study that sevoflurane-mediated renal protection in vivo requires the TGF-β1→SMAD3 signaling pathway.