In recent years, capsid-modified filamentous bacteriophage has become a potential vector for gene delivery into mammalian cells. However, little was known about how the carried gene in the single-stranded genome expressed in mammalian cells. To explore whether the orientation of the carried gene affects its expression in the cells, we prepared EGF-displayed phagemid particles whose genome carried the GFP gene or luciferase gene. The phagemid carried reporter genes either in the same orientation (called (+) gene) or in the contrary orientation (called (-) gene) to filamentous origin. Using these phagemid particles to infect H1299 cells, we found that the phages with (-) reporter genes had about 2-fold transduction efficiency as those with (+) reporter genes. These results indicated that phagemid carrying (-) gene of interest presented a better procedure in phage-mediated gene therapy. Furthermore, camptothecin (CPT) treatment was also applied and found to enhance both kinds of phagemid particles, and (-) gene still produced about 1.5- to 2-fold transduction efficiency compared to those with (+) gene. Thus, it is imperative that we clone the genes of interest in the reverse orientation to filamentous origin to enhance their expressions when preparing phagemid gene delivery vectors. Also, the results suggested