To investigate the effects of dendrobine (DEN) on intestinal barrier repair and insulin resistance improvement, a Caco-2+HT29-MTX/RAW 264.7 co-culture intestinal barrier model and an insulin-resistant HepG2 cell model were established. Barrier function and inflammatory responses were evaluated using transepithelial electrical resistance (TEER), real-time quantitative PCR, and western blot analysis, while glucose metabolism-related indicators were assessed in the insulin resistance model. The results showed that, compared with the model group, DEN maintained TEER values above 200 Ω•cm2 and significantly upregulated the protein expression of tight junction components claudin-1 (72.19%), occludin (82.40%), ZO-1 (75.29%), and mucin MUC2 (70.33%). Correspondingly, mRNA expression levels increased by 1.47%, 452.93%, 2.11%, and 10.38%, respectively. In addition, DEN significantly reduced the levels of pro-inflammatory cytokines TNF-α (14.14%) and IL-6 (68.93%), while markedly increasing the levels of anti-inflammatory cytokines IL-4 (243.96%) and IL-10 (41.35%). In the insulin-resistant HepG2 cell model, DEN at 100, 200, and 300 ng•mL-1 increased glucose uptake in a dose-dependent manner (10.70, 11.83, and 13.04 mmol•L-1, respectively). DEN also enhanced the activities of pyruvate kinase (92.14, 107.09, and 122.02 U•g-1 protein) and hexokinase (29.63, 73.99, and 73.84 U•g-1 protein), and increased intracellular glycogen content by 1.2-, 1.4-, and 1.5-fold, respectively. Overall, DEN effectively repaired intestinal barrier dysfunction and significantly improved insulin resistance. These findings provide a theoretical basis for the development of DEN-based functional foods or therapeutic agents targeting hyperglycemia and metabolic disorders.