Glycerol kinase (Gyk), consisting of 4 isoforms, plays a critical role in metabolism by converting glycerol to glycerol 3‐phosphate in an ATP‐dependent reaction. Only Gyk isoform b is present in whole cells, but its function in the nucleus remains elusive. Previous studies have shown that nuclear orphan receptor subfamily 4 group A member (NR4A)‐1 is an important regulator of hepatic glucose homeostasis and lipid metabolism in adipose tissue. We aimed to elucidate the functional interaction between nuclear Gyk and NR4A1 during hepatic gluconeogenesis in the unfed state and diabetes. We identified nuclear Gyk as a novel corepressor of NR4A1 in the liver; moreover, this recruitment was dependent on the C‐terminal ligand‐binding domain instead of the N‐terminal activation function 1 domain, which interacts with other NR4A1 coregulators. NR4A1 transcriptional activity was inhibited by Gyk via protein‐protein interaction but not enzymatic activity. Moreover, Gyk overexpression suppressed NR4A1 ability to regulate the expression of target genes involved in hepatic gluconeogenesis in vitro and in vivo as well as blood glucose regulation, which was observed in both unfed and diabetic mice. These results highlight the moonlighting function of nuclear Gyk, which was found to act as a coregulator of NR4A1, participating in the regulation of hepatic glucose homeostasis in the unfed state and diabetes.—Miao, L., Yang, Y., Liu, Y., Lai, L., Wang, L., Zhan, Y., Yin, R., Yu, M., Li, C., Yang, X., Ge, C. Glycerol kinase interacts with nuclear receptor NR4A1 and regulates glucose metabolism in the liver. FASEB J. 33, 6736–6747 (2019). http://www.fasebj.org