The causative factors of the vaginitis are not fully understood yet. Epithelial barrier dysfunction plays a critical role in the pathogenesis of vaginitis. This study aims to investigate the role of prolactin (PRL) in the causing the vaginal epithelial barrier dysfunction. Adult rats were treated with water-avoid-stress. The serum levels of PRL were determined by ELISA. T84 cell (T84 cells; a vaginal epithelial cell line) monolayers were prepared to be used assessing the epithelial barrier functions. The expression of occludin in T84 cells was assessed by Chromatin immunoprecipitation assay, methylation specifIc PCR, real time quantitative RT-PCR and Western blotting. The results showed that psychological stress markedly increased the serum levels of PRL in the rat vaginal epithelia. Exposure of T84 cells to PRL in the culture markedly increased the phosphorylation of STAT3 and suppressed the expression of occludin in the cells; the transepithelial electric resistance was decreased and the permeability to a macromolecular tracer was increased in the T84 monolayers, which was mimicked by blocking STAT3, or abolished by over expression of occludin in the epithelial cells. Psychological stress-derived PRL induces vaginal epithelial barrier dysfunction by inhibiting the expression of occludin.