To investigate whether Lactobacillus rhamnosus GG supernatant (LGGs) improved the alcohol-induced barrier damage in Caco-2 cells, Caco-2 cells were used to establish intestinal epithelial cell barrier damage model and detect the integrity and permeability of the intestinal epithelial barrier and bacterial translocation. The effects of LGGs on the expression of tight junction proteins ZO-1, Occludin, and Claudin-1 were analyzed using quantitative reverse transcription polymerase chain reaction (qRTPCR) and western blot. The contents of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) were detected. The changes of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and interleukin-10 (IL-10) were analyzed using qRT-PCR. The results showed that LGGs can prevent alcohol-induced intestinal epithelial cell barrier damage and bacterial translocation. The ZO-1, Occludin, and Claudin-1 mRNA expression levels increased by 42.02%, 45.18%, and 29.84%, respectively, after LGGs intervention. The ZO-1, Occludin, and Claudin-1 protein expression levels increased by 72.52%, 38.72%, and 31.69% respectively. The ROS and MDA contents decreased by 24.92% and 30.19%, respectively. Moreover, the TNF-α, IL-6, and MCP-1 mRNA expression levels were reduced, whereas IL-10 mRNA expression level was elevated. In conclusion, LGGs can attenuate the intestinal barrier damage by enhancing the expression of tight junction proteins, improving oxidative stress damage, and alleviating the inflammatory response. This can provide a theoretical basis for developing functional products against alcoholic liver injury.