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Simple Summary: Simple Summary: The production of eggs is a crucial aspect of the chicken industry. Recent research has indicated that the gut microbiota undergoes dynamic changes and plays a significant role in egg production. However, the relationship between the gut microbiota and host gene expression remains unclear. To investigate this, we collected samples from the intestine (i.e., duodenum, jejunum, and ileum) and its microbiota at different stages of egg-laying (i.e., pre-, peak-, and late-laying) in chickens. Our findings showed that intestinal flora underwent significant changes during different laying periods that included changed genes (i.e., APOA1, APOB, TST, CCDC93, and TMEM175) involved in several transport processes, response to DNA damage, and development of intestinal structure. Additionally, our analysis suggested that specific intestinal microbes were significantly correlated with host gene expression, which indicated an interaction between the gut microbiota and the chicken host. Eggs are a vital dietary component for humans, and it is beneficial to increase egg production to support poultry farming. Initially, the egg production rate rises rapidly with young hens until it reaches its peak, and then it declines gradually. By extending the duration of peak egg production, the hens' performance can be enhanced significantly. Previous studies found dynamic changes in gut microbiota during egg-laying, and several species of microbiota isolated from the chicken gut improved egg-laying performance. However, the interaction between microbes and host gene expression is still unclear. This study provides a more comprehensive understanding of chicken egg-laying by examining dynamic alterations in the microbiota of the entire intestinal tract (i.e., duodenum, jejunum, and ileum) and gene expression. The microbial community in the intestine underwent significant changes during different egg-laying periods (i.e., pre-, peak-, and late-laying periods). Metagenomic functional analysis showed that the relative abundance of biosynthesis of amino acids, secondary metabolites, and cofactors decreased significantly in the duodenum, jejunum, and ileum of aging hens. The relative levels of aldosterone, GnRH, insulin, growth hormone, and other hormone-related pathways increased dramatically in the intestinal microbiota during egg-laying, but only in the microbiota located in the duodenum and ileum. Transcriptome analysis suggested that genes associated with various transport processes were upregulated consistently in the small intestine during egg-laying; genes involved in the development of intestinal structure were down-regulated; and genes involved in response to DNA damage and stress were consistent with changes in laying rate. The abundance of Lactobacillus was related to the expression of ANGPTRL1, ANGPTRL2, ANGPT1L, and NOXO1 in the duodenum; Muricomes was correlated significantly with NFKBIZ, LYG2, and IRG1L expression in the jejunum; and Campylobacter was correlated positively with the expression of KMT2A and USF3 in the ileum. These results indicated that the intestinal microbiota and host gene expression may influence egg production jointly. [ABSTRACT FROM AUTHOR]