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Urban sprawl increasingly affects the ecology of natural populations, including host–microbiota interactions, with observed differences in the gut microbiota between urban and rural hosts. While different mechanisms could explain this pattern, dietary uptake constitutes a likely candidate. To assess the contribution of diet in explaining urban–rural variation in gut microbiota, we performed an aviary experiment in which urban and rural house sparrows were fed with mimics of urban or rural diets. Before the experiment, rural sparrows hosted more diverse gut communities, with a higher relative abundance of Enterococcaceae and Staphylococcaceae and lower abundance of genes involved in xenobiotic degradation and lipid metabolism than their urban counterparts. The experimental diets significantly altered gut microbiota α- and β-diversity and taxonomic composition, with the strongest shifts occurring in individuals …