Hepatitis B X-interacting protein (HBXIP) is able to mediate glucose metabolism reprogramming in breast cancer. To investigate the physiological functions of HBXIP in regulation of glucose metabolism, we generated liver-specific HBXIP conditional knockout C57BL/6 mice using Cre/loxP approach. Liver HBXIP^-/- mice exhibited a phenotype of glycometabolic dysregulation, such as higher fasting blood glucose level, accumulation of hepatic glycogen, recession of blood glucose profiles, and elevation of gluconeogenesis. The expression levels of PEPCK were remarkably up-regulated in the liver tissues of HBXIP^-/- mice. Then, we validated that HBXIP expressions were negatively correlated with those of PEPCK in 30 clinical liver tissues. Mechanistically, luciferase reporter gene assays and ChIP assays showed that HBXIP could inhibit the expression PEPCK at transcription level. Taken together, our findings indicate that HBXIP restrains hepatic gluconeogenesis through suppressing the expression of PEPCK.