Dopamine (DA) is synthesized in gastrointestinal epithelial cells and performs important regulatory effects on the duodenal mucosa. However, the underlying mechanism remains largely unknown. The present study investigated the effect of DA on the duodenal epithelial ion transport in rats by means of short-circuit current (ISC), real-time pH titration, enzyme-linked immunosorbent assay, and immunohistochemistry. The results indicate that basolateral, but not apical, application of DA induced a concentration-dependent ISC downward deflection with an apparent IC50 of 5.34 μmol/L. Basolateral application of dopaminergic receptor D1 (D1) antagonist, SCH-23390, inhibited DA-induced change in ISC (△ISC) in a dose-dependent manner. D1 agonist, SKF38393, mimicked the effect of DA on the ISC. The clear immunoreactivity of D1 subtype D5 (D1b) was at the both apical and basolatoral sides of Brunner's glands and intestinal crypts. Basolateral pretreatment with adenylate cyclase inhibitor, MDL12330A, significantly inhibited DA- and forskolin-induced △ISC. DA and SKF38393 increased the level of intracellular cyclic adenosine monophosphate (cAMP) from 1.55 ± 0.11 to 2.07 ± 0.11 and 5.91 ± 0.25 pmol/L·mg−1, respectively. Furthermore, the serosal DA-induced △ISC was remarkably inhibited by apical administration of K+ channel blockers, Ba2+ and tetraethylammonium, but not by Cl− channel blockers. Serosal DA and D1 agonist did not affect duodenal HCO3− secretion. In conclusion, the present results demonstrate that serosal DA is able to promote rat duodenal epithelial K+ secretion, not HCO3− secretion through D1-mediated and cAMP-dependent pathway. The study provides a new insight in the modulation of DA on the ion transport of duodenal epithelia in rats.