Aim The intestinal barrier is made up of epithelial cells and intercellular junctional complexes to regulate epithelial ion transport and permeability. Dopamine (DA) is able to promote duodenal epithelial ion transport through D1‐like receptors, which includes subtypes of D1 (D1R) and D5 (D5R), but whether D1‐like receptors influence the duodenal permeability is unclear. Methods FITC–dextran permeability, short‐circuit current (ISC), Western blot, immunohistochemistry and ELISA were used in human D5R transgenic mice and hyperendogenous enteric DA (HEnD) rats in this study. Results Dopamine induced a downward deflection in ISC and an increase in FITC–dextran permeability of control rat duodenum, which were inhibited by the D1‐like receptor antagonist, SCH‐23390. However, DA decreased duodenal transepithelial resistance (TER), an effect also reversed by SCH‐23390. A strong immunofluorescence signal for D5R, but not D1R, was observed in the duodenum of control rat. In human D5R knock‐in transgenic mice, duodenal mucosa displayed an increased basal ISC with high FITC–dextran permeability and decreased TER with a lowered expression of tight junction proteins, suggesting attenuated duodenal barrier function in these transgenic mice. D5R knock‐down transgenic mice manifested a decreased basal ISC with lowered FITC–dextran permeability. Moreover, an increased FITC–dextran permeability combined with decreased TER and tight junction protein expression in duodenal mucosa were also observed in HEnD rats. Conclusion This study demonstrates, for the first time, that DA enhances duodenal permeability of control rat via D5R, which provides new experimental and theoretical evidence for the influence of DA on duodenal epithelial barrier function.