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<p>Abstract</p> <p>Background</p> <p>Enhancers are DNA sequences that serve as binding sites for regulatory proteins, and stimulate transcriptional activity independent of their positions and orientations with respect to the transcriptional initiation site. Previous studies considered that baculovirus <it>homologous regions (hrs)</it> function as enhancers in cis. In our study, a plasmid containing homologous region 3 <it>(hr3)</it> enhancer from <it>Bombyx mori nucleopolyhedrovirus (BmNPV) </it>failed to enhance transcription of promoter in other plasmid in co-transfection assays, but strong stimulation occurred when cells were infected by BmNPV. </p> <p>Results</p> <p>The cotransfection results of each BmNPV genomic library plasmid, <it>hr3</it> plasmid and reporter plasmid showed that there were eight library plasmids stimulated the luciferase gene expression remarkably. Sequencing these plasmids revealed that each of them contained the <it>ie-1</it> gene. Transfected plasmids, containing <it>ie-1</it>, <it>hr3</it> and various origin promoter drove reporter gene showed the function was even retained. Cotransfection of <it>hr3</it> functional dissected fragment and<it> ie-1</it> revealed that the 30-bp imperfect palindrome destroyed fragment can't enhance reporter gene expression even though transfected with <it>ie-1</it>.</p> <p>Conclusion</p> <p>IE-1 was the only early factor of BmNPV that could act as a mediator for hr enhancer function in trans and the trans-function was achieved with a broad-spectrum of promoters. The 30-bp imperfect palindrome was the elementary molecular structure by which IE-1 participated in the enhancer function in trans.</p>