With the introduction of next-generation sequencing, a more comprehensive identification of the laying hen gastrointestinal tract microbial population has emerged. There are several factors that can influence the composition and function of the layer hen gastrointestinal tract including age of the bird, diet, and type of feed amendment. The microbial communities in each compartment of the layer hen gastrointestinal tract have been identified and examined for impact on the host. Some compartments such as the ceca harbor a highly complex microbial population of fermentative microorganisms that produce short chain fatty acids. The ceca can also be colonized by foodborne Salmonella and some serovars such as S. Enteritidis can become invasive infecting the reproductive tissues. A variety of feed additives have been applied in attempts to limit Salmonella colonization in laying hens and improve laying hen performance. This chapter will review the function and microbial ecology of the layer hen gastrointestinal tract.