The polyhedrin gene promoter has an essential role in regulating foreign gene expression in baculovirus expression vector systems (BEVS); however, the high-level transcription mechanism is still unknown. One-hybrid screening in yeast is a powerful way of identifying rapidly heterologous transcription factors that can interact with the polyhedrin promoter DNA sequence. In the current study, total RNA was extracted from the fat bodies of fifth-instar silkworm larvae that had been infected with Bombyx mori nuclear polyhedrosis virus (BmNPV) for 5 days; complementary DNA (cDNA) was then generated using reverse-transcription (RT)-PCR to construct a silkworm gene expression library. Key polyhedrin promoter bait sequences were synthesized to generate a bait yeast strain, which was used to screen the one-hybrid cDNA library. In total, 12 positive yeast colonies were obtained from the SD/-Leu/AbA plates; sequencing analysis showed that they belong to two different protein cDNA colonies. Positive colonies underwent bioinformatics analysis, which revealed one colony to be ribosomal proteins [B. mori ribosomal protein SA (BmRPSA)] and the other to be NPV DNA-binding proteins (DBP). To further verify the regulatory function of these two protein groups, transient expression vectors (pSK-IE-dbp and pSK-IE-BmRPSA) were constructed. The recombinant plasmids were then transfected into cultured B. mori N (BmN) cells, which had been infected with a recombinant bacmid containing the gene encoding luciferase (luc). The results showed that overexpression of either dbp or BmRPSA upregulated the polh promoter-driven transcription of luc in BmN cells. In addition, dbp or BmRPSA RNA interference (RNAi) resulted in the downregulation of luciferase reporter expression in BmN cells, demonstrating that DBP and BmRPSA are important for luc transcription. EMSA results further confirmed that DBP could directly bind to the conserved single-stranded polh promoter region in intro. However, EMSA assay also showed that BmRPSA did not bind to this region, precluding a direct DNA association. Both DBP and BmRPSA are important for polh transcription. DBP can regulate polh promoter activity by direct binding to the conserved single-stranded polh promoter region, BmRPSA may regulate polh promoter activity by indirect binding to this region.