To investigate the potential mechanism of hypospadias induced by DEHP in rats to reveal the preventative effect of TGF-β1 in hypospadias induced by DEHP via the reduction of EMT.Time-mated Sprague-Dawley rats underwent cesarean section, and the penises of male pups were collected after exposure to corn oil or DEHP to establish a rat model of hypospadias and to further study the molecular mechanisms of hypospadias in vivo. In addition, the penises were cultured and treated with MEHP or MEHP+TGF-β1 in vitro. Subsequently, histomorphology and elements in TGF-β/Smad signaling pathway changes were evaluated using scanning electron microscopy, immunofluorescence, polymerase chain reaction, and western blot.The development of rat penis and urethral seam fusion were delayed after the treatment with DEHP in vivo or MEHP in vitro compared with the Control group. Moreover, TGF-β1, Smad2/Smad3, and the mesenchymal biomarkers, including α-SMA, N-cadherin, and Vimentin, were decreased. However, the epithelial biomarkers, including E-cadherin, ZO-1, β-catenin, and occludin, were increased. In addition, TGF-β1 could relieve all of the above changes.Gestational DEHP exposure could lead to hypospadias by reducing urethral EMT. Moreover, TGF-β1 could prevent it by regenerating EMT through activating the TGF-β/Smad signal pathway.