Arabidopsis hexokinase1 (HXK1) is a glucose sensor that integrates nutrient and hormone signals to govern gene expression and plant growth in response to environmental cues. How the metabolic enzyme mediates glucose signaling remains a mystery. By coupling proteomic and binary-interaction screens, we discover two nuclear-specific HXK1 unconventional partners: the vacuolar H(+)-ATPase B1 (VHA-B1) and the 19S regulatory particle of proteasome subunit (RPT5B). Remarkably, vha-B1 and rpt5b mutants uniquely share a broad spectrum of glucose response defects with the HXK1 mutant gin2 (glucose-insensitive2). Genetic and chromatin immunoprecipitation analyses suggest that the nuclear HXK1 forms a glucose signaling complex core with VHA-B1 and RPT5B that directly modulates specific target gene transcription independent of glucose metabolism. The findings support a model in which conserved metabolic enzymes and proteins of well-established activities may perform previously unrecognized nuclear functions.