Mice deficient for Gcn2 either globally or specifically in the liver display reduced capacity to maintain glucose homeostasis during fasting suggesting the hypothesis that GCN2 may regulate gluconeogenesis (GNG), which normally plays a key role maintaining peripheral glucose homeostasis. Gcn2 deficient mice exhibit normal insulin sensitivity and plasma insulin, but show reduced gluconeogenesis when administered pyruvate, a gluconeogenic substrate. The basal expression of PEPCK, a rate-limiting enzyme in gluconeogenesis, is abnormally elevated in Gcn2 KO mice in the fed state but fails to be further induced during fasting. The level of TCA cycle intermediates, including malate and oxaloacetate, and the NADH/NAD+ ratio are perturbed in the liver of Gcn2 KO mice either in the fed or fasted state, which may directly impinge upon gluconeogenesis. Additionally, the expression of the CCAAT enhancer binding protein beta (C/EBPβ) in the liver fails to be induced in Gcn2 KO mice after 24hr fasting and the liver-specific Cebpβ KO mice show reduced fasting GNG similar to that seen in Gcn2 deficient mice. Our study demonstrates that GCN2 is important in maintaining GNG in the liver, which is likely to be mediated through regulation of C/EBPβ.