Intestinal mucosa is the interface between the microbial content of the gut and the host's milieu. The goal of this study was to modulate chicken intestinal microflora by in ovo stimulation with galactooligosaccharides (GOS) prebiotic and to demonstrate the molecular responses of the host. The animal trial was performed on meat-type chickens (Ross 308). GOS was delivered by in ovo injection performed into the air cell on day 12 of egg incubation. Analysis of microbial communities and mucosal gene expression was performed at slaughter (day 42 post-hatching). Chyme (for DNA isolation) and intestinal mucosa (for RNA isolation) from four distinct intestinal segments (duodenum, jejunum, ileum, and caecum) was sampled. The relative abundance of Bifidobacterium spp. and Lactobacillus spp. in DNA isolated from chyme samples was determined using qPCR. On the host side, the mRNA expression of 13 genes grouped into two panels was analysed with RT-qPCR. Panel (1) included genes related to intestinal innate immune responses (IL-1[beta], IL-10 and IL-12p40, AvBD1 and CATHL2). Panel (2) contained genes involved in intestinal barrier function (MUC6, CLDN1 and TJAP1) and nutrients sensing (FFAR2 and FFAR4, GLUT1, GLUT2 and GLUT5). GOS increased the relative abundance of Bifidobacterium in caecum (from 1.3% to 3.9%). Distinct effects of GOS on gene expression were manifested in jejunum and caecum. Cytokine genes (IL-1[beta], IL-10 and IL-12p40) were up-regulated in the jejunum and caecum of the GOS-treated group. Host defence peptides (AvBD1 and CATHL2) were up-regulated in the caecum of the GOS-treated group. Free fatty acid receptors (FFAR2 and FFAR4) were up-regulated in all three compartments of the intestine (except the duodenum). Glucose transporters were down-regulated in duodenum (GLUT2 and GLUT5) but up-regulated in the hindgut (GLUT1 and GLUT2). In conclusion, GOS delivered in ovo had a bifidogenic effect in adult chickens. It also modulated gene expression related to intestinal immune responses, gut barrier function, and nutrient sensing.