Background: The declines in both laying performance and egg shell quality during late production period have adverse effects on long production cycle. Improving nutrition of laying hens is a crucial measure to reverse the declination. Selenium (Se) plays important roles in antioxidant defense, redox balance, immune response, and modulation of gut microbiota. However, the mechanism underlying selenium yeast regulating the interaction between transcriptome and gut microbiota to influence laying performance, is still unclear. Here, we use the transcriptome and 16S rRNA analysis to investigate how selenium yeast alters the gene expression and microbiota composition of ileum in aged laying hens.Results: In this study, selenium yeast ameliorated the depression in aged laying performance with a significant increase of laying rate in 0.30 mg/kg group. Furthermore, functional enrichment and STEM analysis were performed using RNA-Seq, which indicated selenium yeast activated metabolic progresses (e.g. Glycerolipid metabolism, Glycerophospholipid metabolism, and fatty acid metabolism), immune response and oxidative stress response. Four hub genes (TXNRD1, DLD, ILK and LZTS2) were involved in intestinal metabolism which was closely associated with Se deposition/status. Additionally, Se increased the abundance of beneficial bacteria including Veillonella, Turicibacter, and Lactobacillus whiledecreasing the abundance of pathogenic bacteria Stenotrophomonas by 16S rRNA-Seq. The Integrated analysis of omics revealed that several microbiotas (Maritalea, Alteromonas, Geobacter, etc.) were positively associated with both Se content and laying rate, and there was a markable correlation between several specific microbiotas (Aliivibrio, Anaerobacillus, Shewanella, etc.) and the immune response pathways (regulation of acute inflammatory response, positive regulation of lymphocyte activation and IFN gamma response). Meanwhile, the “switched on” gene PSCA had a positive relationship with Veillonella, and a negative relationship with the opportunistic pathogens Stenotrophomonas. CCA analysis indicated that both the Se content and the laying rate were highly positively correlated with Anaerobacillus, Alteromonas, Loktanella and the positive regulation of lymphocyte activation, but were negative correlated with Streptococcus, Devosia, Aerococcus, Intestinibacter and fatty acid metabolic progress. Conclusions: Overall, our study provides unprecedented insights showing that selenium yeast supplementation can affect immune response, metabolic processes, and specific microbiota composition to ameliorate the egg production deterioration in aged laying hens.