Probiotics benefit the health and production performance of chickens, but their impact on egg and eggshell quality, particularly in the later stage, remains unclear. Here, 1-day-old Tianfu green shell-laying hens were fed either non-probiotics feed (n = 180) or feed supplemented with 100 mg / kg probiotics (n = 180). 16S rDNA sequencing indicated that dietary probiotics decreased the distribution of uterine p_Firmicutes, g_Fusobacterium, and s_Fusobacterium_unclassified, while increased p_Proteobacteria, g_Ralstonia, and s_Ralstonia_unclassified. PICRUSt2 and Bug Base analysis revealed enrichment in fatty acid metabolism, thiamine metabolism, vitamin B6 metabolism pathways and increased relative abundance of Proteobacteria, Firmicutes, Bacteroidetes. With LDA >
4.5, 35 and 25 marker bacterial taxa were identified in the uterus and cecum, respectively. Probiotics significantly increased uterine villi length and width, and the expressions of ATP2B2,SLC26A9,TF,OC-17,OCX-32, and OVAL in the uterus at the early and peak laying stage. Meantime, probiotics improved egg quality, pore density of eggshell barrier layer, and levels of Ca