Cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key glucogenic enzyme, is controlled at the transcriptional level. Our objective was to determine regulatory elements within the bovine PCK1 promoter that control transcription in response to cyclic AMP (cAMP), glucocorticoids, and propionate (PROP). Putative DNA promoter transcription factor protein binding sequences were identified for cAMP response element (CRE) at −94 to −87 and for Hepatic Nuclear Factor 4α (HNF4α) at +68 to +72 (HNF4α1) and −1078 to −1074(HNF4α2). To test control of transcription, the wild-type (WT) bovine PCK1 promoter (−1238 to +221) was ligated to a luciferase reporter gene and transfected into H4IIE cells followed by incubation with 2.5 mM PROP, 1 mM cAMP (cAMP), 5 μM dexamethasone (DEX) or their combinations. The functionality of CRE, HNF4α1, and HNF4α2 cis-regulatory elements was determined using deletion mutations of the core transcription factor binding regions within the PCK1 promoter DNA. The deletion mutations tested were HNF4α1⁻; HNF4α2⁻; CRE⁻; HNF4α1⁻/HNF4α2⁻; CRE⁻/HNF4α1⁻; CRE⁻/HNF4α2⁻; and CRE⁻/HNF4α1⁻/HNF4α2⁻. H4IIE cells were transfected with the promoter-reporter constructs and exposed to treatments for 23 h. Luciferase activity was measured in the cell lysates as a direct proxy for bovine PCK1 promoter activity. Within each construct, treatment effect was expressed as the fold change of luciferase activity relative to base media control (n = 3 cell preparations). Analyses of variance of the data were performed using the Proc Mixed procedure of SAS 9.3. Exposure to cAMP, DEX, cAMP+DEX, PROP, cAMP+PROP, cAMP+DEX+PROP induced (P < 0.05) expression of the WT promoter relative to no addition controls by 2.0, 2.3, 3.9, 6.0, 7.3, 14.4 ± 1.4 x respectively. A similar pattern was observed for each single mutant bovine PCK1 promoter. Responses to cAMP, DEX, PROP and their combinations were abolished for mutations lacking both HNF4α1 and CRE binding sites indicating that these elements act synergistically to control bovine PCK1 transcription.