Enterotoxigenic Escherichia coli (ETEC) is a significant cause of infectious diarrhea in animals. In this study, yeast surface display technology was employed to investigate the effects of ETEC enterotoxin fusion protein on the intestinal flora and mucosal immunity of rats. ETEC estA, estB, and eltAB (heat-labile and heat-stable toxins) were expressed on the surface of yeast. Rats were divided into normal saline, yeast and display yeast groups. Fecal and jejunal content samples were collected on the 7th, 14th, and 21st days. Rats were then fed ETEC for 3 days before again collecting these samples. Levels of SIgA, IL-2, IL-4, IFN-gamma, and microbial population density and diversity were documented by ELISA, T-RFLP and real-time PCR. The results demonstrated that estA, estB, and eltAB fusion proteins were expressed on the surface of yeast. Following ETEC challenge, levels of SIgA, IL-2, IL-4, IFN-gamma, and, the numbers and variety of intestinal microbes were significantly increased in rats receiving display yeast and yeast. These factors were significantly decreased in rats given normal saline and yeast. Our results indicate that display yeast and yeast can increase the number and diversity of intestinal microbes in rats and improve intestinal immune function. After ETEC challenge, the display yeast can better maintain the balance of intestinal bacteria and mucosal immunity.