Trillions of microorganisms that inhabit the intestinal tract form a diverse and intricate ecosystem with a deeply embedded symbiotic relationship with their hosts. As more detailed information on gut microbiota complexity and functional diversity accumulates, we are learning more about how diet-microbiota interactions can influence the immune system within and outside the gut and host health in general. Heat shock proteins are a set of highly conserved proteins that are present in all types of cells, from microbes to mammals. These proteins carry out crucial intracellular housekeeping functions and unexpected extracellular immuno-regulatory features in order to maintain the mucosal barrier integrity and gut homeostasis. It is becoming evident that the enteric microbiota is one of the major determinants of heat shock protein production in intestinal epithelial cells. This review will focus on the interactions between diet, gut microbiota and their role for regulating heat shock protein production and, furthermore, how these interactions influence the immune system and the integrity of the mucosal barrier.