Low glucose stimulated phosphorylation of pregnane X receptor (PXR) at Ser350 in correlation with an increased gluconeogenesis in human hepatoma-derived HepG2 cells. Only glucose, but neither insulin nor glucagon, stimulated this phosphorylation. Here, serine/threonine kinase, vaccinia related kinase 1 (VRK1)-mediated phosphorylation of PXR is now defined as this glucose-elicited novel signal. In low glucose conditions, VRK1 directly phosphorylates PXR at Ser350, enabling PO3-PXR to scaffold protein phosphatase PP2Cα. This PP2Cα dephosphorylates serine/threonine kinase 2 (SGK2) at Thr193. This dephosphorylation dissociates SGK2 from and actives the phosphoenolpyruvate carboxykinase 1 (PCK1) gene as phosphorylated SGK2 binds and represses the gene. Conversely, VRK1 self-represses its activity to phosphorylate PXR through cyclin-dependent kinase 2 (CDK2) in high glucose conditions, resulting in the repression of the PCK1 gene. This PXR phosphorylation was also observed in fasting mouse livers. Thus, the VRK1-CDK2-PXR-PP2Cα-SGK2 pathway can be a novel physiological cell signaling that regulates gluconeogenesis in response to glucose.