The role of intestinal epithelial cells (IECs) in the physiology of the gastrointestinal tract (GIT) of chickens and pathogenesis of various diseases in chickens is still poorly understood. IECs line the GIT and represent the border between the unsterile environment and the sterile internal tissues. Bacterial, viral, fungal, or parasitic pathogens are able to invade or pass IECs under certain circumstances and cause various diseases. Pathogen-host interactions in the chicken gut are poorly understood because of the lack of suitable in vitro and ex vivo models. In this context, there is a need to optimize the cell isolation and culture conditions to be able to provide reproducible IEC cultures with defined epithelial characteristics. We compared different mechanical IEC isolation protocols and cell culture media and established a reproducible primary intestinal epithelial cell culture model from specific-pathogen-free layer-type chickens. By using isolated crypts from the duodenum of 5- to 12-wk-old birds to create the starting material, we were able to culture replicating cells between 7 and 10 days. Cells built an almost closed monolayer and showed epithelial-like characteristics, such as the expression of cytokeratin and epithelial cadherin. The primary IEC cultures described in this study represent a suitable model with which to investigate in vitro pathogen-host interactions relevant to the chicken gut.