Background: The aim was to develop a better experimental model which could facilitate further studies assessing the vertical HCV gene transmission via human spermatozoa, and verify the possibility of father-to-child transmission of the HCV gene.Methods: The recombinant plasmid plRES2-EGFP-HCV C was constructed.Fluorescence in situ hybridization was performed to detect the integration of the HCV C gene in human sperm genome and in zygotes pronucleus.Results: Successful construction of recombinant plasmid plRES2-EGFP-HCV C was confirmed by restriction mapping, PCR and sequencing.Positive HCV C DNA signals were observed in sperm heads, human sperm chromosomes and two-cell embryos in transfected samples.No positive signal was found in normal control and HCV infected groups.Conclusions: The recombinant plasmid plRES2-EGFP-HCV C was successfully constructed.The HCV C gene was able to pass through sperm membrane and integrate into the sperm genome.Human sperm carrying the HCV C gene was able to achieve normal fertilization.The replication of the sperm-mediated HCV C gene was synchronized with that of the host genome.Our results provide direct evidence for vertical transmission of the HCV C gene from father-to-child via human sperm.