The aim of this study was to analyze the influence of quercetin on intestinal barrier function using the human colonic epithelial cell line HT-29/B6 and rat small and large intestine in vitro. Rat native ileum and late distal colon were incubated in Ussing chambers, and the total resistance (RT) was measured, and expression of tight junction proteins was characterized in immunoblots. By simulating inflammatory conditions with TNF-α, we examined the barrier-preventive effects of quercetin. Incubation with TNF-α led to a decrease of RT in HT-29/B6 cell monolayers, which could be partially inhibited by quercetin. In accordance with cell culture experiments, quercetin increased mucosal resistance of rat ileum and late distal colon. Thus, barrier disturbance in late distal colon specimens induced by TNF-α and IFN-γ could be partially prevented by coincubation with quercetin. These findings demonstrate that quercetin enhances barrier function in rat small and large intestine and possesses protective effects on cytokine-induced barrier damage.