As a novel fermented beverage, seabuckthorn vinegar drink (SVD) has demonstrated promising efficacy in promoting intestinal health. However, its specific mechanism of action remains unclear and requires further investigation. Based on the characterization of basic physicochemical properties and antioxidant activity of SVD, this study further explored its regulatory effects on gene expression in intestinal epithelial cells using transcriptome sequencing analysis. The results showed that SVD had a pH of 3.75, total acid content of 1.02 g/100 mL, total phenolics of 88.97 mg/100 mL, total flavonoids of 237.14 mg/100 mL, and a centrifugal sedimentation rate of 3.64%, exhibiting strong ABTS?? and DPPH? scavenging activities. In the Caco-2 cell model, SVD regulated the mRNA expression levels of AQP3、AQP4 and MUC2 genes. Preliminary western blotting results suggested an increasing trend in the protein expression of Claudin-1 and Occludin. Transcriptome sequencing analysis revealed that SVD regulated the differential expression of 2,818 genes in Caco-2 cells, with differently expressed genes enriched in signaling pathways such as Notch, Hippo, and MAPK. These findings preliminarily suggest that SVD has the potential to regulate intestinal water transport and maintain intestinal barrier function by modulating the expression of genes related to aquaporins and mucins in intestinal epithelial cells. This study provides a preliminary theoretical basis for the application of SVD as a functional food in the field of intestinal health.