Nuclear ubiquitous casein and cyclin-dependent kinase substrate (NUCKS) is highly expressed in the brain and peripheral metabolic organs, and regulates transcription of a number of genes involved in insulin signalling. Whole-body depletion of NUCKS (NKO) in mice leads to obesity, glucose intolerance and insulin resistance. However, a tissue-specific contribution of NUCKS to the observed phenotypes remains unknown. Considering the pivotal roles of insulin signalling in the brain, especially in the hypothalamus, we examined the functions of hypothalamic NUCKS in the regulation of peripheral glucose metabolism. Insulin signalling in the hypothalamus was impaired in the NKO mice when insulin was delivered through intracerebroventricular injection. To validate the hypothalamic specificity, we crossed transgenic mice expressing Cre-recombinase under the Nkx2.1 promoter with floxed NUCKS mice to generate mice with hypothalamus-specific deletion of NUCKS (HNKO). We fed the HNKO and littermate control mice with a normal chow diet (NCD) and a high-fat diet (HFD), and assessed glucose tolerance, insulin tolerance and metabolic parameters. HNKO mice showed mild glucose intolerance under an NCD, but exacerbated obesity and insulin resistance phenotypes under an HFD. In addition, NUCKS regulated levels of insulin receptor in the brain. Unlike HNKO mice, mice with immune-cell-specific deletion of NUCKS (VNKO) did not develop obesity or insulin-resistant phenotypes under an HFD. These studies indicate that hypothalamic NUCKS plays an essential role in regulating glucose homoeostasis and insulin signalling in vivo.