Chicken infection with Salmonella enterica, subspecies enterica serovar Typhimurium (S. Typhimurium) is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the cecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Our objectif was to establish ileal and cecal organoids by seeding intestinal epithelial stem cells in a 3-dimensional (3D) culture environment reproducing in vitro the stem cell niche to investigate the pathophysiology of Salmonella-avian intestinal epithelium interactions. These organoids form a monolayer of polarized epithelial cells containing the diverse differentiated epithelial cells, recapitulating the original structure and function of the native epithelium. Using this 3D avian organoid model, we were able to visualize and compare the ability of S. Typhimurium to invade and intracellularly replicate, according to the chicken intestinal segment, as well as its impact on the proliferation and differentiation of cells constituting the intestinal epithelium by transcriptomic analysis. This new approach allows a more representative study of avian intestinal epithelium-Salmonella interactions and an efficient reduction of the use of live animals for experimentation.