||The maintenance of metabolic homeostasis presents a major challenge to animal biology. Dysregulation of this process is central to the etiology of diabetes and obesity, which have placed an ever-increasing burden on global human health. Nuclear receptors are a family of ligand-regulated transcription factors that play important roles in preventing metabolic dysfunction by coordinating downstream transcriptional programs to support physiological homeostasis. This thesis is focused on one of these nuclear receptors, Hepatocyte Nuclear Factor 4A (HNF4A), which acts as a critical regulator of systemic glucose homeostasis. Mutations in HNF4A were established 20 years ago as the monogenic cause of an inherited form of diabetes called Maturity-Onset Diabetes of the Young 1 (MODY1). The mechanistic link between HNF4A dysfunction and diabetes onset, however, remains unclear. In this dissertation, I present work that provides new insights into the mechanisms by which HNF4 supports physiological homeostasis and prevents diabetes. The fruit fly Drosophila melanogaster provides an ideal system for studies of metabolism and physiology that can guide our understanding of these pathways in mammals. We developed and optimized methods for studies of metabolism in Drosophila to further advance its utility in these efforts. Using these techniques, I investigated potentially conserved roles for Drosophila HNF4 (dHNF4) in supporting development and metabolic homeostasis. Through this work, I discovered that dHNF4 mutants recapitulate hallmark features of MODY1, including hyperglycemia and impaired glucose-stimulated insulin secretion (GSIS). dHNF4 functions in the adult insulin producing cells and the fat body to support GSIS and peripheral glucose clearance during adulthood. These functions are linked to a role for dHNF4 in promoting mitochondrial activity in the mature animal through up-regulation of both nuclear and mitochondrial-encoded genes involved in oxidative phosphorylation (OXPHOS). By extending my analysis to mammals through a collaborative effort with several labs, we uncovered evidence suggesting that the role for HNF4 in supporting mitochondrial function has been conserved through evolution. Taken together, my work has identified HNF4 as an important regulator of mitochondrial activity, and suggests that this function has been conserved through evolution to support normal development, physiology, and metabolic homeostasis.