Cancer immunotherapy has attracted much attention in recent years because of the ability of immune system to identify tumor cells and limit their growth. Icariin (ICA) is a natural flavonoid glucoside isolated from Epimedium plants and has shown a variety of pharmacological activities such as anti-inflammatory effects, immunological regulation and anticancer potency. Furthermore, it has immunoadjuvant effects on enhancing Th1-immune response, suggesting that ICA may serve as an adjuvant for cancer immunotherapy. In this study, we used P815 mouse mastocytoma tumor model and immunized them with P815AB peptide and/or ICA. Our results demonstrated that ICA could increase the cytotoxic T lymphocytes (CTL) response for P815AB peptide on the tumor-bearing DBA/2J mice. In addition, the percentage of CD4⁺CD8⁺/CD3⁺CD69⁺/CD69⁺NKG2D⁺ positive cells in splenocytes of the tumor-bearing mice all significantly increased after combined immunization with ICA and P815AB peptide. This illustrated that ICA could enhance the immunogenicity of P815AB and improve the ability of T cells and CTLs in recognizing the tumor cells. Moreover, ICA improved the function of peritoneal macrophages with effects of inhibition on tumor growth. Besides, we discussed the possible mechanism of ICA to enhance body immunity by detecting the expression level of MHC-I and related genes in B16-F10 and RMA/S cells. The results suggested that ICA has the potential to up-regulate LMP/TAP related molecules and induce the expression of MHC-I, which increase the immune surveillance and keep cancer in remission. In conclusion, ICA showed an anti-tumor effect both in vitro and in vivo and may be an effective antigen adjuvant for cancer treatment by enhancing tumor-specific immunity.