The human gut microbiota is integrally involved in the metabolism of nutrients contained within the human diet. Studies into human nutrition have primarily been carried out using human and animal models. These studies are extremely important in our understanding of human nutrition, however, suffer from inherent limitations including unique microbial compositions between individuals, compliance in human studies, inability to carry out mechanistic studies, and inability to interrogate proximal regions of the gut without applying invasive techniques. In vitro gut simulator systems circumvent many of these limitations in animal and human models by allowing control of gut environmental conditions, decreasing variability observed between subjects, and enabling mechanistic investigations and interrogations of inaccessible regions of the gut. In this work a custom biofermentation system, the human gut simulator, was designed, validated, utilizing previously reported gut conditions, capable of temperature, pH, and atmosphere regulation, nutrient transit, and it allows real-time sampling of vessel contents or addition of exogenous agents. The human gut simulator was further employed to the study of gut microbiota response to dietary long chain fatty acids as a sole nutrient source, following stabilization on a rich `western’ medium. Microbiota showed rapid responses to the transition from western to fat medium; where a lack of carbohydrates and proteins resulted in decreased community density. Specific members of the microbiota were capable of utilizing long chain fatty acids, including Bilophila, Alistipes, and Escherichia/Shigella. Interestingly, members of the microbiota incapable of metabolizing long chain fatty acids included beneficial microbes Roseburia, Bifidobacterium, and Akkermansia. Ordination and principal response curves analyses highlighted a significant effect of medium change on shifts in microbial composition over time. In conjunction with in vitro studies, human volunteers were enrolled to assess responses of microbiota to diets high in proteins, carbohydrates, or fats. Microarray analysis revealed specific individual host responses to test diets with smaller community wide effects. Increasing the amount of protein in the diet had a positive impact on relative abundance of Akkermansia, Alistipes, Enterococcus, and Lactococcus, while higher carbohydrates and fats resulted in higher abundances of Bifidobacterium, and Alistipes and Escherichia/Shigella, respectively. Together these results indicate that the Human Gut Simulator allows for robust studies of the human gut microbiota, and offers a foundation for conducting nutritional interventions in human subjects.