Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that β2 → 1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2 → 1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 β2 → 1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2 → 1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain β2 → 1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain β2 → 1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2 → 1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that β2 → 1-fructans are specific ligands for TLR2. To conclude, β2 → 1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2 → 1-fructan-mediated health effects.