The gene for glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis, is expressed in a tissue-specific manner in the liver and kidney. To understand the molecular mechanisms regulating liver-specific expression of the G6Pase gene, we characterized G6Pase promoter activity by transient expression assays. The G6Pase promoter is active in HepG2 hepatoma cells, but inactive in JEG3 choriocarcinoma or 3T3 cells. DNA elements essential for optimal and liver-specific expression of the G6Pase gene were contained within nucleotides −234 to +3. Deletion analysis revealed that the G6Pase promoter contained three activation elements (AEs) at nucleotides −234 to −212 (AE-I), −146 to −125 (AE-II), and −124 to −71 (AE-III). AE-I contains binding sites for hepatocyte nuclear factors (HNF) 1 and 4. Electromobility shift and cotransfection assays demonstrated that HNF1α, but not HNF4, bound to its cognate site and transactivated G6Pase gene expression. The G6Pase promoter contained five HNF3 motifs, 1 (−180/−174), 2 (−139/−133), 3 (−91/−85), 4 (−81/−75), and 5 (−72/−66), and all five sites bound HNF3γ with high affinity. Transient expression and cotransfection assays showed that HNF3 site 1 is not required for basal promoter activity, but is essential for HNF3γ-activated transcription from the G6Pase promoter. We further showed that HNF3 sites 3, 4, and 5 were essential for basal G6Pase promoter activity and transactivation by HNF3γ. AE-II contains, in addition to a HNF3 motif, a cAMP-response element (CRE) and a C/EBP half-site. The G6Pase(−146/−116) DNA containing AE-II formed multiple protein−DNA complexes with HepG2 nuclear extracts, including HNF3γ, CRE-binding protein (CREB), C/EBPα, and C/EBPβ. We showed that AE-II mediated transcription activation of the G6Pase gene by cAMP.