DNA immunization is a promising approach in generating immune responses to infectious pathogens in many different animal models. In an effort to augment the anti‐[hepatitis C virus (HCV) core] immune response, generated after DNA immunization, the importance of vaccination regimen regarding dose and boosting was investigated in the present study. Balb/c mice were intramuscularly injected with an expression plasmid encoding a truncated variant comprising amino acids 1–176 of the HCV core protein. The highest anti‐core antibody titres (1:3700) were detected in mice inoculated with 50–100 μg of core‐encoding plasmid. Additionally, we demonstrated that antibody levels induced by a single injection of DNA could be further increased by boosting with a second injection of DNA three weeks after primary immunization. However, administration of additional doses or lengthening of the resting period between inoculations resulted in similar or even weaker anti‐core antibody responses. A similar anti‐(HCV core) lymphoproliferative response was also detected in animals that had the highest level of anti‐core antibodies. These results indicate that, in clinical trials, vaccination regimen might be a critical factor in generating optimal anti‐(HCV core) immune responses after genetic immunization.