The luminal-mucosal interface of the intestinal tract is the first relevant location where microorganism-derived antigens and all other potentially immunogenic particles face the scrutiny of the powerful mammalian immune system. Upon regular functioning conditions, the intestinal barrier is able to effectively prevent most environmental and external antigens to interact openly with the numerous and versatile elements that compose the mucosal-associated immune system. This evolutionary super system is capable of processing an astonishing amount of antigens and non-immunogenic particles, approximately 100 tons in one individual lifetime, only considering food-derived components. Most important, to develop oral tolerance and proper active immune responses needed to prevent disease and inflammation, this giant immunogenic load has to be managed in a way that physiological inflammatory balance is constantly preserved. Adequate functioning of the intestinal barrier involves local and distant regulatory networks integrating the so-called brain-gut axis. Along this complex axis both brain and gut structures participate in the processing and execution of response signals to external and internal changes coming from the digestive tract, using multidirectional pathways to communicate. Dysfunction of brain-gut axis facilitates malfunctioning of the intestinal barrier, and vice versa, increasing the risk of uncontrolled immunological reactions that may trigger mucosal and brain low-grade inflammation, a putative first step to the initiation of more permanent gut disorders. In this chapter, we describe the structure, function and interactions of intestinal barrier, microbiota and brain-gut axis in both healthy and pathological conditions.