Aim: To identify a shuttle promoter that can mediate gene expression in both insect cells and mammalian cells to facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. Methods: Recombinant baculoviruses carrying the β-galactosidase reporter gene under the control of an early to late (ETL) promoter of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) or a cytomegalovirus immediate early promoter (CMV promoter) were constructed. COS1, HeLa, CHO-K1, hFob1.19, and MCF-7 mammalian cells were tested for the expression of β-galactosidase. Results: ETL promoter activity was higher in bone-derived hFob1.19 than in COS1, HeLa, CHO-K1, or MCF-7 mammalian cells. The transient plasmid transfection assay indicated that ETL promoter activity in mammalian cells was dependent on baculovirus gene expression. Conclusion: ETL promoter activity in mammalian cells is baculovirus gene expression-dependent, and the shuttle promoter will facilitate the application of baculovirus expression vectors in mammalian cell expression systems and for gene therapy.