<b><i>Background:</i></b> In peripheral tissues, the lipid droplet (LD) organelle links lipid metabolism, inflammation, and insulin resistance. Little is known about the brain LDs. <b><i>Objectives:</i></b> We hypothesized that hypothalamic LDs would be altered in metabolic diseases. <b><i>Methods:</i></b> We used immunofluorescence labeling of the specific LD protein, PLIN2, as the approach to visualize and quantify LDs. <b><i>Results:</i></b> LDs were abundant in the hypothalamic third ventricle wall layer with similar heterogeneous distributions between control mice and humans. The LD content was enhanced by high-fat diet (HFD) in both wild-type and in low-density lipoprotein receptor deficient (<i>Ldlr</i> –/– HFD) mice. Strikingly, we observed a lower LD amount in type 2 diabetes mellitus (T2DM) patients when compared with non-T2DM patients. <b><i>Conclusions:</i></b> LDs accumulate in the normal hypothalamus, with similar distributions in human and mouse. Moreover, metabolic diseases differently modify LD content in mouse and human. Our results suggest that hypothalamic LD accumulation is an important target to the study of metabolism.