Abstract: Erythropoietin-producing hepatocellular carcinoma A3 (EphA3) is a member of the largest subfamily of tyrosine kinase receptors—Eph receptors. Previous studies have shown that EphA3 is associated with tissue development. Recently, we have found that the expression of EphA3 is elevated in the hypothalamus of mice with diet-induced obesity (DIO). However, the role of EphA3 in hypothalamic-controlled energy metabolism remains unclear. In the current study, we demonstrated that the deletion of EphA3 in the hypothalamus by CRISPR/Cas9-mediated gene editing promotes obesity in male mice with high-fat diet feeding rather than those with normal chow diet feeding. Moreover, the deletion of hypothalamic EphA3 promotes high-fat DIO by increasing food intake and reducing energy expenditure. Knockdown of EphA3 leads to smaller intracellular vesicles in GT1-7 cells. The current study reveals that hypothalamic EphA3 plays important roles in promoting DIO.