Ubiquitin-specific protease (USP) 2 participates in glucose metabolism in peripheral tissues such as the liver and skeletal muscle. However, the glucoregulatory role of USP2 in the central nervous system is not well known. In this study, we focus on USP2 in the ventromedial hypothalamus (VMH), which has dominant control over systemic glucose homeostasis. ISH using a Usp2 -specific probe showed that Usp2 mRNA is present in VMH neurons, as well as other glucoregulatory nuclei, in the hypothalamus of male mice. Administration of a USP2-selective inhibitor, ML364 (20 ng/head), into the VMH elicited a rapid increase in the circulating glucose level in male mice, suggesting USP2 has a suppressive role on glucose mobilization. ML364 treatment also increased serum norepinephrine concentration, while it negligibly affected serum levels of insulin and corticosterone. ML364 perturbated mitochondrial oxidative phosphorylation in neural SH-SY5Y cells, and subsequently promoted the phosphorylation of AMP-activated prote...