Bacteriophages are appropriate gene carriers that might be targeted toward target cells using different strategies. Here we prepared a transferrin-targeted M13-based gene nanocarrier (Tf-targeted M13-GFP) and examined its gene delivery and expression efficacy in the AGS cell line. M13 phagemid particles bearing a GFP expression cassette (M13-GFP) were obtained from a recombinant lambda phage through an in vivo excision procedure. Chemical coupling of human holotransferrin molecules (Tf) to the surface of these phagemid particles resulted in Tf-targeted M13-GFP formation, which was then characterized by Phage-ELISA and Cell-ELISA experiments. Immunocytochemistry (ICC) and fluorescence-activated cell sorting (FACS) analysis were used for internalization assay and examination of gene delivery/expression efficacies in the human AGS cell line. The ELISA experiments revealed high-density attachment of Tf molecules to the surface of M13-GFP particles and ICC confirmed highly efficient internalization of the Tf-targeted M13-GFP particles into the AGS cells. Moreover, FACS analysis showed significant increase of GFP-positive cell counts in the samples treated with Tf-targeted M13-GFP (8.09%) in comparison with the samples treated with wild M13-GFP (1.2%). We conclude that this strategy might improve phage-mediated gene delivery and expression in eukaryote cells.