Abstract: Objective To study the effect of hypoxia on cofilin activation in intestinal epithelial cells and its relation with distribution of tight junction protein zonula occludens 1 (ZO-1). Methods The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer cells. The monolayer-cell specimens were divided into control group (no treatment), hypoxic group (exposed to hypoxia), and normoxic group (exposed to normoxia) according to the random number table. Western blotting was used to detect the protein expressions of cofilin and phosphorylated cofilin (p-cofilin) of cells in normoxic group and hypoxic group exposed to normoxia or hypoxia for 1, 2, 6, 12, and 24 h and control group, with 9 samples in control group and 9 samples at each time point in the other two groups. The other monolayer-cell specimens were divided into hypoxic group (exposed to hypoxia) and control group (no treatment) according to the random number table. Cells in hypoxic group exposed to hypoxia for 1, 2, 6, 12, and 24 h and control group were obtained. Morphology and distribution of F-actin was observed with laser scanning confocal microscopy, the ratio of F-actin to G-actin was determined by fluorescence method, and distribution of ZO-1 and cellular morphology were observed with laser scanning confocal microscopy. The sample number of last 3 experiments was respectively 3, 6, and 3 in both hypoxic group (at each time point) and control group. Data were processed with paired t test, analysis of variance of repeated measurement, and LSD-t test. Results The protein expressions of cofilin and p-cofilin of cells between normoxic group exposed to normoxia for 1 to 24 h and control group showed no significant changes (with t_cofilin values from -0.385 to 1.701, t_p-cofilin values from 0.040 to 1.538, P values above 0.05). There were no obvious differences in protein expressions of cofilin of cells between hypoxic group exposed to hypoxia for 1 to 24 h and control group (with t values from 1.032 to 2.390, P values above 0.05). Compared with that in control group, the protein expressions of p-cofilin of cells were greatly reduced in hypoxic group exposed to hypoxia for 1 to 24 h (with t values from 4.563 to 22.678, P values below 0.01), especially exposed to hypoxia for 24 h. The protein expressions of cofilin of cells between normoxic group and hypoxic group at each time point were close (with t values from -0.904 to 1.433, P values above 0.05). In hypoxic group, the protein expressions of p-cofilin of cells exposed to hypoxia for 1, 2, 6, 12, and 24 h were 0.87±0.08, 0.78±0.05, 0.89±0.07, 0.68±0.07, and 0.57±0.06, respectively, significantly lower than those in normoxic group (0.90±0.07, 0.97±0.06, 1.00±0.06, 1.00±0.05, and 0.99±0.05, with t values from 3.193 to 16.434, P values below 0.01). In control group, F-actin in the cytoplasm was abundant, most of it was in bunches. The trend of F-actin was disorderly in hypoxic group from being exposed to hypoxia for 1 h, shortened in length or even dissipated. The ratios of F-actin to G-actin of cells in hypoxic group exposed to hypoxia for 12 and 24 h (0.89±0.12 and 0.84±0.19) were obviously decreased as compared with that in control group (1.00, with t values respectively 3.622 and 3.577, P values below 0.01). There were no obvious differences in the ratios of F-actin to G-actin of cells between hypoxic group exposed to hypoxia for 1, 2, and 6 h and control group (with t values from 0.447 to 1.526, P values above 0.05). In control group, cells were compact in arrangement, and ZO-1 was distributed continuously along the cytomembrane. From being exposed to hypoxia for 2 h, cells became irregular in shape in hypoxic group. ZO-1 was distributed in discontinuous fashion along the cytomembrane with breakage in hypoxic group exposed to hypoxia for 24 h. Conclusions Hypoxia may cause the disorder of dynamic balance between F-actin and G-actin by inducing cofilin activation, which in turn leads to the changes in distribution of tight junction protein ZO-1 in intestinal epithelial cells.