Pancreatic β-cell-type-specific expression of the insulin gene requires both ubiquitous and cell-enriched activators, which are organized within the enhancer region into a network of protein-protein and protein-DNA interactions to promote transcriptional synergy. Protein-protein-mediated communication between DNA-bound activators and the RNA polymerase II transcriptional machinery is inhibited by the adenovirus E1A protein as a result of E1A’s binding to the p300 coactivator. E1A disrupts signaling between the non-DNA-binding p300 protein and the basic helix-loop-helix DNA-binding factors of insulin’s E-element activator (i.e., the islet-enriched BETA2 and generally distributed E47 proteins), as well as a distinct but unidentified enhancer factor. In the present report, we show that E1A binding to p300 prevents activation by insulin’s β-cell-enriched PDX-1 activator. p300 interacts directly with the N-terminal region of the PDX-1 homeodomain protein, which contains conserved amino acid sequences essential for activation. The unique combination of PDX-1, BETA2, E47, and p300 was shown to promote synergistic activation from a transfected insulin enhancer-driven reporter construct in non-β cells, a process inhibited by E1A. In addition, E1A inhibited the level of PDX-1 and BETA2 complex formation in β cells. These results indicate that E1A inhibits insulin gene transcription by preventing communication between the p300 coactivator and key DNA-bound activators, like PDX-1 and BETA2:E47.