Intestinal epithelial barrier (IEB) damage is an important aspect in inflammatory bowel disease (IBD). The objective of this study was to explore the protective effects and mechanisms of arabinogalactan (AG) on lipopolysaccharide (LPS)-stimulated IEB dysfunction. The results show that AG (1, 2, and 5 mg/mL) mitigated 100 μg/mL LPS-stimulated IEB dysfunction through increasing transepithelial electrical resistance (TEER), reducing fluorescein isothiocyanate (FITC)–dextran (4 kDa) flux, and up-regulating the protein and mRNA expression of tight junction (TJ) proteins (Claudin-1, Zonula occludens-1 (ZO-1) and Occludin). In addition, AG ameliorated LPS-stimulated IEB dysfunction by reducing interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β levels, decreasing the reactive oxygen species (ROS) level, increasing superoxide dismutase (SOD) activity, increasing the glutathione (GSH) level, and decreasing the levels of malondialdehyde (MDA) and intracellular calcium ([Ca2+]i). Furthermore, 2 mg/mL AG up-regulated the expression of silent information regulator 1 (SIRT1), the phosphorylated adenosine monophosphate-activated protein kinase (AMPK), and peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and inhibited the phosphorylation of nuclear factor kappa-B (NF-κB) and the inhibitor of NF-κBα (IκBα). Therefore, AG could maintain IEB integrity by activating AMPK/SIRT1 and inhibiting the NF-κB signaling pathway. In conclusion, AG can regulate the AMPK/SIRT1/NF-κB signaling pathway to reduce inflammation and oxidative stress, thus alleviating LPS-stimulated IEB damage.