•Polysaccharide PCP-II was first used as adjuvant in rabies vaccine.•PCP-2 could induce rapid and high antibody responses in mice and dogs.•PCP-II could activate lymphocytes and promote cytokines secretion in mice.•PCP-II is a good candidate for adjuvant on inactivated rabies vaccine for animal and human use. Adjuvants are important components of vaccination strategies because they boost and accelerate the immune response. The aim of this study was to investigate the adjuvant activity of PCP-II, a polysaccharide isolated from Poria cocos, together with an inactivated rabies vaccine. The polysaccharide PCP-II was compared with the common veterinary rabies vaccine adjuvant Alhydrogel by co-administration of either adjuvant with the inactivated rabies virus rCVS-11-G to mice via the intramuscular route. Blood samples were collected to determine the virus-neutralizing antibody (VNA) titer and assess activation of B and T lymphocytes. Inguinal lymph node samples were collected, and proliferation of B lymphocytes was measured. Splenocytes were isolated, and antigen-specific cellular immune responses were evaluated by enzyme-linked immunospot and immunosorbent assays (ELISpot assay and ELISA, respectively). The results showed that PCP-II enhanced and promoted an increase in the VNA titer in the mice compared to Alhydrogel. Flow cytometry assays revealed that the polysaccharide activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood. Assessment of antigen-specific cellular immune responses showed that PCP-II strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes. All of these data suggest that PCP-II possesses excellent adjuvant activity and enhances both cellular and humoral immunity in mice. After examining the adjuvant activities of PCP-II in mice, dogs were immunized with rCVS-11-G together with Alhydrogel or PCP-II as an adjuvant; the control group was injected with a commercial rabies vaccine. Serum samples were collected, and the VNA titers were measured. PCP-II caused increases in the VNA titers in both the booster and single-dose immunization tests when co-administered with rCVS-11-G compared with Alhydrogel. The VNA titer of the commercial vaccine group was also significantly lower than that of the PCP-II group. These data indicate that PCP-II is an excellent candidate adjuvant for inactive rabies vaccines in the veterinary setting.