Despite interest in understanding glucagon-like peptide-1 (GLP-1) production, the factors important for GLP-1 biosynthesis remain poorly understood. We examined control of human proglucagon gene expression in NCI-H716 cells, a cell line that secretes GLP-1 in a regulated manner. Insulin, phorbol myristate acetate, or forskolin, known regulators of rodent proglucagon gene expression, had no effect, whereas sodium butyrate decreased levels of NCI-H716 proglucagon mRNA transcripts. The inhibitory effect of sodium butyrate was mimicked by trichostatin A but was not detected with sodium acetate or isobutyrate. The actions of butyrate were not diminished by the ERK1/2 inhibitor PD98059, p38 inhibitor SB203580, or soluble guanylate cyclase inhibitor LY83583 or following treatment of cells with KT5823, a selective inhibitor of cGMP-dependent protein kinase. NCI-H716 cells expressed multiple proglucagon gene transcription factors including isl-1, pax-6, pax-2, cdx-2/3, pax-4, hepatocyte nuclear factor (HNF)-3α, HNF-3β, HNF-3γ, and Nkx2.2. Nevertheless, the butyrate-dependent inhibition of proglucagon gene expression was not associated with coordinate changes in transcription factor expression and both the human and rat transfected proglucagon promoters were transcriptionally inactive in NCI-H716 cells. Hence, NCI-H716 cells may not be a physiologically optimal model for studies of human enteroendocrine proglucagon gene transcription.