Owing to their immune-modulatory action on the intestinal mucosa immune cells, the n-3 and n-6 polyunsaturated fatty acids (PUFA) have been suggested to modulate the risk and development of inflammatory bowel diseases. Failure in the intestinal barrier is an important hallmark of inflammatory bowel diseases. This study aimed at evaluating the impact of dietary PUFA on tight junction protein localisation and on the modulation of epithelial permeability under physiological conditions or under an inflammatory stress. For this purpose, we first confirmed the accumulation of PUFA in phospholipid fractions of Caco-2 cells upon 7 days of incubation with specific PUFA. Thereafter, Caco-2 cells were cultured in inserts, which provide a model of the human intestinal barrier. Accumulation of dietary n-3 PUFA in phospholipids did not affect the presence of occludin in tight junction complexes, while that of dietary n-6 PUFA decreased it. Whatever the PUFA, at 30 μM, no distortion of the Caco-2 barrier function was observed. Otherwise, 150 μM of docosahexaenoic acid (DHA) affected ZO-1 intensity under normal conditions, but not occludin or the barrier function parameters. Finally, to simulate an inflammatory state, cells were exposed for 24 h to interleukin-1β, tumor necrosis factor-α, interferon-γ at their basolateral side and to lypopolysaccharides at both sides. DHA limited the effect of inflammatory stimulus on occludin, ZO-1 and barrier function. In conclusion, this study has evidenced the specific effect of individual PUFA to modulate occludin and ZO-1 localization, according to the inflammatory status of this in vitro model of the intestinal barrier.