The complex interaction between the metazoan host and its commensal gut microbiota is one of the essential features of symbiosis in the animal kingdom. As there is a burgeoning interest to decipher the molecular dialog that shapes host-microbiota mutualism, the use of gnotobiotic model organism becomes an imperative approach to unambiguously parse the specific contributions to such interaction from the microbiome. In this review, we focus on several remarkable gnotobiotic studies in Drosophila that functionally depicted how the gut microbes can alter host physiology and behavior through transcriptomic regulation, hormonal control, and diet modification. These results in concert illustrate that the gnotobiotic flies mono- or poly-associated with members of its gut microbiota deliver a versatile and powerful model that is amenable to different types of studies ranging from classic genetics to large-scale systems approaches.