Polyphenols and phenolic acids make up the majority of the complex mixture of compounds, such as grape seed extract (GSE). In this study, the protective effects and working mechanisms of Red Grape Seed extract (GSE) against ethanol (EtOH)-induced tight junction (TJ) and adherent junction protein dysfunction were investigated in the rats’ liver, kidney, and spleen. Twenty-four adult Albino Sprague Dawley rats were used and divided into 4 groups: Group 1 (Control Negative), and Group 2 (Control Positive), the rats received a twice-weekly dosage of 40% ethanol; In Group 3, rats were given Red GSE only; Group 4 (treatment group), rats were given EtOH + Red GSE, which was administered three times weekly orally through gavage tube for six weeks. The liver, kidney, and spleen tissue were processed for the H&E staining and Claudin-1, Claudin-5, and E-cadherin biomarkers by IHC analysis. Treatment with Red GSE reduced the histopathologic abnormality scores in the liver (7 vs. 13), kidney (2 vs. 8), and spleen (3 vs. 6) when compared to the EtOH group, which alleviated the harmful effects of EtOH. Additionally, Red GSE increases the expression of Claudin-1, Claudin-5, and E-cadherin by a membrane pattern in the parenchyma of the liver, kidneys, and spleen, mostly by moderate-strong immunostaining in contrast to EtOH, which displayed weak staining in membranous and cytoplasmic regions. Red GSE preserves the tight junction in the damaged cells and is a promising natural agent against ethanol-induced barrier dysfunction.