Abstract： Objective To investigate the change of tissue structure and epithelial barrier function of excluded esophagus after esophageal exclusion surgery for tracheoesophageal fistula (TEF). Methods Twentyeight hybrid dogs were divided into 3 groups with random number method. For group A (n=12), fistula was excluded; for group B (n=12), only the esophagus was excluded; and group C (n=4) was the control group. The normal and excluded esophagus of the three groups were observed through general sampling, HE staining, transmission electron microscope (TEM), scanning electron microscope (SEM) and Lanthanum trace labeling. The interstitial space dilation (ISD) of the cells on the esophageal mucosa was measured and the number of desmosome in the intercellular space was calculated. Results The changes of tissue structure and epithelial barrier function of the excluded esophagus were similar between group A and group B. Compared with group C, group A and B showed the following changes: esophageal structure changed, submucous glandular tissues reduced or disappeared; the interstitial space increased, the number of desmosome decreased, and the epithelial barrier function attenuated, but the basal lamina did not changed and lanthanum did not break through it. For the dogs in group C, esophageal mucosa could be observed, submucosa was complete and a large number of gland tissues and micrangiums could be seen in it. The number of desmosome for group A, B and C was 0.21±0.03 entries/μm2, 0.22±0.05 entries/μm2, and 0.32±0.03 entries/μm2 respectively, which showed a significant difference between group A, B and group C (Plt;0.05), while there was no significant difference between group A and group B (Plt;0.05). The average interstitial space for group A, B and C was 2.11±0.56 μm, 2.04±0.77 μm, and 1.84±0.49 μm respectively. There was no significant difference between group A and B, while there was significant difference between group A, B and group C. Conclusion The esophageal exclusion surgery for refractory TEF is safe, feasible, and effective with few complications, and deserved to be popularized.