Aims/hypothesisNF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated howRELAimpacts the beta cell transcriptional landscape and provides network control over glucoregulation.MethodsWe generated novel mouse lines harbouring beta cell-specific deletion of either theRelagene, encoding the canonical NF-κB transcription factor p65 (βp65KO mice), or theIkbkggene, encoding the NF-κB essential modulator NEMO (βNEMOKO mice), as well as βA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator geneTnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation–sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme.ResultsReladeficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However,Reladeletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as βp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance requiredRelabut was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout ofIkbkg(NEMO), or beta cell overexpression ofTnfaip3(A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genesSlc2a2,Capn9andPfkmidentified within the large network of islet enhancer hub genes showed dysregulated expression in βp65KO islets.Conclusions/interpretationThese data demonstrate an unappreciated role forRELAas a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes.Graphical AbstractSupplementary InformationThe online version of this article (10.1007/s00125-023-05931-6) contains peer-reviewed but unedited supplementary material.