Hepatitis C virus (HCV) F protein has been identified for more than ten years, but its functions remain unclear. In order to understand its roles in viral replication and infection, in this study 5 stop codons (nt406T-A, nt433T-A, nt472G-A, nt479/481G-A, G-A, nt613C-A) were introduced in plasmid J6JFH1 to construct J6JFH1/ΔF which interrupt F protein expression, and then monitor the viral RNA replication and viral protein expression. Our data showed that the five mutations did not affect core gene replication and expression. The virus strain J6JFH1/ΔF significantly reduced in the expression of viral proteins in transfected cells compared with wild-type J6JFH1, and viral RNA levels dropped by nearly 95% (J6JFH1/ΔF (7.39×10⁶±2.54×10⁴) vs wild type (1.17×10⁸±1.46×10⁷), P < 0.001). The infectivity of virus released into the supernatant significantly reduced similarly. Further study found that these five mutations changed secondary structure of HCV core gene. In order to further explore the underlying reasons for the weakening of J6JFH1/ΔF virus replication and infection, we constructed five separate mutant viruses based on J6JFH1, respectively, and form J6JFH1m1 nt406T-A, J6JFH1m2 nt433T-A, J6JFH1m3 nt472G-A, J6JFH1m4 nt479/481G-A, G-A, J6JFH1m5 nt613C-A. These 5 virus mutants do not influence the secondary structure of the core region, while J6JFH1m1, J6JFH1m2, J6JFH1m3, J6JFH1m4, J6JFH1m5 respectively stopped F protein expression shifting from nt374-383, nt417-419, nt436-445, nt474-477, nt597-605, in which J6JFH1m5 stopped all reported forms of F protein. By in vitro transcription and transfection into Huh7.5.1 cells, 48 hours post-transfection, the detection of intracellular viral protein NS5A expression levels, viral RNA in transfected cells, and virus titers in supernatant were carried out. The results showed that no significant differences were found among groups five separate mutants and wild type. These results indicated that the lack of F protein itself did not affect HCV viral replication and virion assembly and infectivity. All the results were seemed to result from the secondary structure of the core gene, but the mechanism was yet to be further studied. We concluded that HCV F protein deficiency did not affect translation of viral replication and packaging and release of virus particles, the core gene secondary structure changes had a great impact on viral replication and protein expression.