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Tight junction protein is representative regulator of gut permeability. Also, it has been noted for controlling inflammatory responses through tight junction. Therefore, in this study, we examined that whether tight junction protein is changed in aged mice, and to further, confirmed the effect of treadmill exercise on the tight junction protein. In in vitro study, doxorubicin that induces cell senescence was treated to Caco2 cells (colon cell) to mimic aging effect. After that, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), exercise mimic chemical that stimulates AMPK level, was also administered to Caco2 cells. In animal study, 2 months and 21 months C57BL/6 J mouse were treated with treadmill exercise for 4 weeks (YE = 5, OE = 5). Then, the tight junction protein expression level was examined by western blot. Also, serum lipopolysaccharide (LPS) and zonulin level were analyzed to identify gut permeability. In vitro studies showed that doxorubicin downregulates tight junction protein expression levels in Caco2 cell, and also AICAR treatment upregulates tight junction protein expression levels. In animal study, 4 weeks treadmill exercise upregulated claudin-1 (p 0.05) and occludin (p 0.01) protein expression level in 21 months old mice. Also, zonula occluden-1 (ZO-1) protein expression level was not significant difference among all mice group. In addition, old mice group had higher level of serum LPS compared to young mice group, but the level was downregulated in both 2 months and 21 months mice group after four weeks of treadmill exercise. Zonulin, which is known as degrading tight junction protein, is not significantly changed by both age and exercise. This study compared that tight junction protein expression level in old mice compared to its level in young mice, and also clarified that the effect of treadmill exercise on tight junction protein in both young and old mice.