Objective To investigate the effect of Pleurotus ferulae polysaccharide (PFP) as an adjuvant on the infertility induced by canine zona pellucida 3 (CZP3) DNA vaccine (pcDNA3-CZP3). Methods Female mice were intramuscularly immunized with pcDNA3-CZP3 alone or co-immunized with pcDNA3-CZP3 and aluminum adjuvant or PFP for three times. CZP3-specific antibody titers of antiserum were determined by ELISA. The maturation of dendritic cells (DCs) and the proliferation of T lymphocytes were detected by flow cytometry. The litter of the immunized mice were counted. Results Compared with pcDNA3-CZP3 alone, pcDNA3-CZP3 combined with aluminum or PFP significantly increased the CZP3-specific antibody titers. Moreover, PFP as an adjuvant significantly up-regulated the expressions of CD86 and MHCII on DCs, enhanced the proliferation of T cells, and decreased the fertility rate and mean litter. Conclusion PFP can enhance the infertility efficacy of CZP3 DNA vaccine through increasing the humoral and cellular immune responses, promoting DC maturation and T cell proliferation.