The expression of genes encoding key hepatic gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), is regulated at the transcriptional level by a network of transcription factors and cofactors, including cAMP response element-binding protein (CREB). It has been suggested that increased endoplasmic reticulum (ER) stress in the liver impairs hepatic glucose metabolism. However, the direct effect of ER stress on hepatic gluconeogenesis is still not clear. Here, we investigated whether ER stress influences hepatic gluconeogenesis and whether this process is mediated by activating transcription factor 6 (ATF6) through the inhibition of cAMP-mediated activation of CREB. A cAMP stimulant, forskolin, and 8-bromoadenosine-cAMP increased PEPCK and G6Pase mRNA expression in H4IIE rat hepatoma cells, and ER stress induced by tunicamycin or thapsigargin decreased the expression of these genes in forskolin or 8-bromoadenosine-cAMP-treated cells. In a transient transfection study, ATF6 inhibited the PEPCK and G6Pase promoters. Also, adenovirus-mediated overexpression of ATF6 in H4IIE cells decreased forskolin-stimulated PEPCK and G6Pase gene expression. Moreover, the inhibition of endogenous ATF6 expression by small interfering RNAs restored the ER stress-induced suppression of PEPCK and G6Pase gene expression. Transient transfection of ATF6 inhibited transactivation by CREB on the PEPCK and G6Pase promoters, and a gel shift assay showed that Ad-ATF6 inhibits forskolin-stimulated CREB DNA-binding activity. Finally, we found that expression of ATF6 decreased fasting-induced PEPCK, G6Pase mRNA expression, and blood glucose levels in mice. Taken together, these data extend our understanding of ER stress and the regulation of liver gluconeogenesis by ATF6.