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The traditional Chinese medicine Artemisia annua can prevent and treat hepatitis following an unclear mechanism. The aim of this study was to evaluate the effects of A. annua polysaccharides (AAP) on hepatitis C virus (HCV). A pcDNA3.1/NS3 expression vector was constructed. Ninety female BALB/c mice were randomly divided into six groups: high-dose AAP (1 mg/mL) + HCV/NS3 plasmid; middle-dose AAP (0.5 mg/mL) + HCV/NS3 plasmid; low-dose AAP (0.1 mg/mL) + HCV/NS3 plasmid; HCV/NS3 plasmid; high-dose AAP (1 mg/mL); normal saline control (N = 15). Except the control group and the high-dose AAP group, other groups were inoculated with 50 μg pcDNA3.1-HCV/NS3 plasmid. Serum antigenic-specific antibody was detected after the last immunization, and the levels of secreted IFN-γ and IL-4 were measured. pcDNA3.1/NS3 plasmid was successfully constructed, and the extracted product contained HCV/NS3 sequence. Compared with single inoculation with HCV/NS3 DNA vaccine, the specific antibody levels induced by middle-dose AAP plus HCV/NS3 DNA vaccine were significantly different in weeks 1, 3 and 5 (P0.05). However, there were no significant differences in the antibody levels induced by high-dose and low-dose AAP as adjuvant compared with those of single inoculation with DNA vaccine (P0.05). The level of serum IFN-γ secretion was significantly higher than that of IL-4 secretion. Compared with the single HCV/NS3 DNA vaccine group, AAP plus HCV/NS3 DNA vaccine groups had significant increased IFN-γ levels (P0.05), but the IL-4 levels were not significantly different among these groups (P0.05). AAP, as the adjuvant of HCV/NS3 DNA vaccine, can widely regulate the humoral immunity and cellular immune function of normal and cyclophosphamide-induced immunocompromised mice. AAP can promote IFN-γ secretion probably by inducing Th1-type cellular immune response.