One 13-week-old Xiangjia Yellow Chicken No. 2 was used as the experimental animal. Single-cell RNA sequencing(scRNA-seq) technology was applied to dissect cell types of three small intestinal segments, including the duodenum, jejunum and ileum, at the cellular level, thereby systematically constructing a single-cell atlas of chicken small intestine. Based on this atlas, we further performed functional enrichment analysis, cell-cell communication inference and pseudotime analysis of epithelial cells. The results showed that the epithelial mucosal layer of chicken small intestine was composed of twelve distinct cell types, including B cells, tuft cells, dendritic cells, endothelial cells, enteroendocrine cells, erythrocytes, goblet cells, proliferating cells, fibroblast, T cells, plasma cells and enterocytes. The proportions of each cell type varied across distinct small intestinal segments. As the core functional cells responsible for nutrient absorption, enterocytes exhibited a relatively higher proportion in the duodenum, suggesting that the duodenum served as the primary site for nutrient absorption within the small intestine. Immune cell populations including T cells, B cells and plasma cells were abundant in all three small intestinal segments, demonstrating that intestinal immune responsed exert critical effects on nutrient metabolism and host-microbe interactions in the chicken small intestine. The functional enrichment analysis of differentially expressed genes in each cell type was consistent with the cell annotation results. Strong ligand-receptor communication signals were detected between enteroendocrine cells and tuft cells, indicating that these two cell types may play a vital role in maintaining the homeostasis of small intestinal mucosal epithelium. The enterocytes could be further categorized into 8 subpopulations, including intestinal stem cells, APOB+ enterocytes, FABP1/2+ enterocytes, immune-associates enterocytes, ESRRG+ enterocytes, MZB1+ enterocytes, APOA1+ enterocytes, and BEST4+ enterocytes. Genes including ESRRG, FABP1, FABP2, PTPRF and FTH1 were identified as key regulators involved in enterocyte differentiation. Immune-associated enterocytes, APOB+ enterocytes, MZB1+ enterocytes, and FABP1/2+ enterocytes may be associated with necrotizing enterocolitis.