In this study, the effect and mechanisms of curdlan sulfate (CS3) on hepatitis B virus (HBV) infection and promoting immune response of the mice immunized with recombinant hepatitis B surface protein (HBsAg) were investigated. The results showed that CS3 could inhibit HBV infection of HepG2 and HepaRG cells, especially the process of HBV particle binding to the cell surfaces. The surface plasmon response (SPR) technology indicated that CS3 could bind with recombinant HBsAg and the binding ability depended on the content of sulfate groups on the polysaccharide chains. Co-administration of CS3 to BALB/c mice immunized with HBsAg significantly enhanced the influx of macrophages and dendritic cells in spleen, increased antigen-specific CD4+ and CD8+ cell numbers, and promoted splenocyte proliferation. The titer of HBsAg-specific antibodies was also augmented by use of CS3 as a vaccine adjuvant. The higher expression of interferon (IFN)-γ, lower expression of interleukin (IL)-4, and higher IgG2a/IgG1 ratio within the anti-HBsAg antibodies in mice immunized with HBsAg plus CS3 than those in mice receiving HBsAg alone indicated that CS3 induced a shift toward a Th1-biased immune response. These results presented that CS3 could be developed as an immunotherapy agent or vaccine adjuvant for HBV infection treatment or prevention.