Objective To evaluate the effect of the galacto-oligosaccharides (GOS) on the intestinal mucosal tight junction and barrier function of rats with induced cold restraint stress. Methods Forty Wistar rats were randomly divided into four groups: two cold resistant stress (CRS) groups fed with either a standard diet (CRS group) or 4 g/kg GOS per day (CRS+GOS group) and two sham CRS groups fed with a standard diet (control group) or 4 g/kg GOS per day (GOS group). All the four groups were fed for a total of 10 days, and the CRS and CRS+GOS groups were subjected to cold restraint stress on the 10^th day. Then the blood samples of all the four groups were collected via cardio puncture for determination of serum diamine oxidas (DAO) activity; finally all the rats were sacrificed and the intestinal tissues were collected for pathological and electronic microscopic examination, with the occludin mRNA level measured by real-time PCR and the occludin protein expression by immune-histological method. Results Compared with the control, the serum DAO levels of CRS group and the CRS+GOS group were significantly increased(P<0.01); and that in the CRS group was significantly higher than that in the CRS+GOS group (P<0.01). The levels of occludin mRNA and protein expression were significantly decreased in the two CRS groups (P<0.01), and that in the CRS group was significantly lower than that in the CRS+GOS group (P<0.01); the serum DAO levels, the expressions of occludin mRNA and protein were not significantly different between the control and the GOS groups. Conclusion Feeding with GOS can decrease plasma level of DAO in rats, increase expression of intestinal occludin mRNA and protein in rat ileum, indicating that GOS can improve the mucosal tight junction and intestinal barrier function.