The use of probiotics in poultry production has increased rapidly, and this movement has been promoted by global events, such as the prohibition or decline in the use of antibiotic growth promotants in poultry feeds. There has been a persistent search for alternative feed additives, and probiotics have shown that they can restore the composition of the gut microbiota, and produce health benefits to the host, including improvements in performance. Probiotics have shown potential to increase productivity in poultry, especially in flocks challenged by stressors. However, the outcomes of probiotic use have not always been consistent. There is an increasing demand for well defined products that can be applied strategically, and currently, probiotic research is focusing on delineating their mechanisms of action in the gut that contribute to an improved efficacy. In particular, mechanisms involved in the maintenance and protection of intestinal barrier integrity and the role of the gut microbiota are being extensively investigated. It has been shown that probiotics modulate intestinal immune pathways both directly and through interactions with the gut microbiota. These interactions are key to maintaining gut homeostasis and function, and improving feed efficiency. Research has demonstrated that probiotics execute their effects through multiple mechanisms. The present review describes recent advances in probiotic use in poultry. It focuses on the current understanding of gut homeostasis and gut health in chickens, and how it can be assessed and improved through supplementation of poultry diets with probiotics in poultry diets. In particular, cellular and molecular mechanisms involved in the maintenance and protection of gut barrier structure and function are described. It also highlights important factors that influence probiotic efficacy and bird performance.