Classically, erythropoietin (EPO) is known as the master regulator of erythropoiesis. However, EPO has recently emerged as a pleiotropic cytokine with non-hematopoietic roles, which includes cytoprotection, cell proliferation and regulation of energy metabolism. Deletion of the EPOR, which disrupts EPO signaling in non-erythroid tissues, via knockout of the EPO receptor, promotes metabolic dysregulation (e.g., obesity, glucose intolerance, changes in whole- body substrate selection). The kidney is well recognized as the major source of EPO; however, non-renal sources of EPO have been identified. Therefore, the role of non-renal sources of EPO remains to be elucidated, as does the role of EPO in regulating energy metabolism. As endothelial cells are pervasive in all tissues and stabilize metabolic regulators (e.g., hypoxia- inducible factor 1) in response to both hypoxia-dependent and -independent stressors, the endothelium is well suited for metabolic regulation. Thus, the aim of the investigation was to determine the physiological significance of endothelial-derived EPO. We hypothesized that the endothelium is a source of EPO that functions in a paracrine fashion to maintain energy homeostasis. To investigate the physiological relevance of endothelial-derived EPO on energy homeostasis we generated an endothelial cell-specific deletion of EPO by crossing EPO-LoxP mice with mice expressing Cre recombinase under the control of the mouse endothelial-specific promoter tyrosine kinase (Tek)(EPOfl/fl-ENDO). We assessed the deletion of endothelial EPO on circulating EPO levels, exercise capacity, in vitro muscle function (e.g. force/ frequency and fatigability) and metabolic parameters (e.g. glycogen content, whole-body substrate selection). We have identified a transgenic mouse model that exhibits constitutive endothelial-specific deletion of the EPO gene. The EPOfl/fl-ENDO mice followed Mendelian genetics and had normal hematocrit, body weight and cardiac function as compared to EPOWT. However, deletion of endothelial-derived EPO resulted in exercise intolerance that was not a result of impairments in intrinsic muscle function (e.g., force and fatigability). EPOfl/fl-ENDO mice exhibited reduced glycogen content in glycolytic, but not oxidative muscles, and increased glycogen content in the liver. The modification in local carbohydrate metabolism did not result in changes to whole- body substrate metabolism measured by indirect calorimetry at rest. EPOfl/fl-ENDO mice did exhibit reductions in glycolytic skeletal muscle capillary: fiber ratio that may play a role in the reduced exercise tolerance, as a result of a potential for impaired oxygen delivery. This thesis demonstrates that the endothelium is a non-renal source of physiologically relevant EPO that is necessary for maintenance of carbohydrate metabolism.