The present study was conducted to unravel the molecular mechanism underlying the synergistic effect of capsaicin and quercetin on the regulation of glucose metabolism in small intestine. The Caco-2 monolayer model was established to investigate the effect of capsaicin and quercetin， both singly and combined， on the cell viability and cell membrane permeability. Additionally， western blotting was utilized to determine the expression levels of Occludin， cytoplasmic protein ZO-1， epidermal growth factor receptor (EGFR) and fibrillar muscle protein F-actin as well as key proteins involved in glucose transport including GLUT2， SGLT1 and Na+/K+-ATPase. Results showed that the cell viability of all treatment groups was higher than 75%， demonstrating no toxic effect of capsaicin and quercetin on Caco-2 cells. Administration of capsaicin and quercetin increased the TEER values of Caco-2 monolayer model (315.70±26.65) Ω·cm2， which indicated smaller permeability and thus stronger barrier function. Furthermore， compared to the CK group， capsaicin and quercetin treatments upregulated the expression levels of Occludin (>0.65)， ZO-1 (>0.51)， EGFR (>0.19)， and F-actin (>0.04)， but downregulated that of GLUT2 (>1.00)， SGLT1 (>0.78) and Na+/K+-ATPase (>0.87). In summary， capsaicin and quercetin can exert a synergistic effect on the regulation of glucose metabolism in small intestine and high dose levels of capsaicin and quercetin at a ratio of 1∶3 was found to be optimal. The potential molecular mechanism can be attributed to the enhancement of barrier function of small intestine and downregulation of key proteins involved in glucose transport， which consequently hinders glucose absorption to achieve a hypoglycemic effect.