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 that CPT could enhance both the replication of single-stranded DNA and its transcription.