Oestrogens regulate body weight through their action on hypothalamus to modulate food intake and energy expenditure. Hypothalamic de novo ceramide synthesis plays a central role on obesity induced by oestrogen deficiency. Depletion in oestrogens is also known to be associated with glucose intolerance, which favours type 2 diabetes (T2D). However, the implication of hypothalamic ceramide in the regulation of glucose homeostasis by oestrogen is unknown. Here, we studied glucose homeostasis and insulin secretion in ovariectomized (OVX) female rats. OVX induces body weight gain associated with a hypothalamic inflammation and impaired glucose homeostasis. Genetic blockade of ceramide synthesis in the ventromedial nucleus of the hypothalamus (VMH) reverses hypothalamic inflammation and partly restored glucose tolerance induced by OVX. Furthermore, glucose-stimulated insulin secretion (GSIS) is increased in OVX rats due to a raise of insulin secretion second phase, a characteristic of early stage of T2D. In contrast, GSIS from isolated islets of OVX rats is totally blunted. Inhibition of ceramide synthesis in the VMH restores GSIS from isolated OVX islets and represses the second phase of insulin secretion. Stimulation of oestrogen receptor α (ERα) by oestradiol (E2) down-regulates ceramide synthesis in hypothalamic neuronal GT1-7 cells but no in microglial SIM-A9 cells. In contrast, genetic inactivation of ERα in VMH upregulates ceramide synthesis. These results indicate that hypothalamic neuronal de novo ceramide synthesis triggers the OVX-dependent impairment of glucose homeostasis which is partly mediated by a dysregulation of GSIS.