Introduction: Dendritic cells (DCs) have been recently employed as carriers for vaccines against several viral infections. The present study was designed to develop a prophylactic vaccine against hepatitis C virus (HCV) using DCs treated with Berberis vulgaris root extract (BRE), as a preclinical study. Methods: BRE was prepared and injected to female BALB/c mice for DCs expansion. The expanded splenocyte cells (EDC) were purified and efficiently loaded ex vivo with HCV-nonstructural protein 3, NS3, (EDC-NS3). Mice were subcutaneously inoculated with EDC-NS3 vaccine candidate thrice with 4-week-intervals and IL-12, IFN-γ, IL-4, IL-10, MHC class II, CD3, CD16, indoleamine 2, 3 dioxygenase (IDO) and total protein levels were measured, post-vaccination by PCR and flow cytometry. Moreover, cytotoxic T lymphocyte and humoral immune responses were examined. Results: Our data revealed that immunization with EDC-NS3 vaccine elevated IL-12, IFN-γ and IL-4 expressions as well as MHC II and CD16 at protein levels. It also elicited strong HCV-NS3-specific humoral and cellular immune responses. However, the expressions of CD3, IDO, and IL-10 were down-regulated, post-vaccination. Conclusion: EDC-NS3 immunization serves as an innovative modality for immunoprophylaxis against HCV infection.