Supplying novel feed ingredients for pig production is crucial to enhance food security and decrease the environmental impact of meat production. Several studies have focused on evaluating the beneficial health effects of yeast in pigs. However, its use as a protein source has been partially addressed. Previously, we have shown that yeast at high inclusion levels maintains growth performance and digestibility, while nutrient digestibility, intestinal villi height and fecal consistency were improved. The present study combined microbiome, short-chain fatty acid, and immune parameter analysis to investigate the effect of high inclusion of yeast in diets for post-weaning piglets. Our results showed that yeast did not have a significant impact on the hematological or biochemical parameters in blood. The different immune cell subpopulations isolated from blood and distal jejunal lymph nodes (DJLN) were analyzed by flow cytometry and showed that yeast diet induced an increased number of the subtype of leukocytes CD45+/CD3-/CD8+, a special type of Natural Killer (NK) cells. Also, a very mild to moderate infiltration of neutrophilic granulocytes and lower IgA level were observed in the colon of yeast fed piglets. The microbiome profiling in different compartments of the gastrointestinal tract of piglets was performed using 16S rRNA metabarcoding. The results showed that 40% replacement of dietary protein had a statistically significant effect on the microbial communities in cecum and colon, while the microbial population in ileum and jejunum were not affected. Analysis of predicted microbial metabolic pathways analysis revealed significant upregulation of short-chain fatty acids, ether lipid metabolisms, secondary bile acids, and several other important biosynthesis pathways in cecum and colon of pigs fed yeast. In conclusion, the results showed that diet containing 40% of yeast protein positively shaped microbial community in the large intestine and increased the number of a specific subpopulation of NK cells in the DJLN. These results showed that yeast modulates the microbiome and decreases the secretion of IgA in the colon of post-weaning pigs.