With no specific antiviral drugs and preventive vaccines against Mpox virus (MPXV),the epidemic has led to the declaration of a Public Health Emergency of International Concern.As a developmental direction for new vaccines, studies of subunit vaccines based upon MPXVantigen proteins are lacking. In this study, A29L, M1R, A35R, and B6R of MPXV were expressed and purified from a rokaryotic system. The four MPXV antigen proteins in combination were mixed with aluminum hydroxide or CpG7909 as adjuvant, and subsequently used to inoculate mice. The results of enzyme-linked immunosorbent assay (ELISA), flow cytometry analyses, and enzyme-linked immunospot (ELISPOT) assays indicated that A29L, M1R, A35R, and B6R elicited high-level antigen-specific antibodies and CD4+ T cells-based cellular immune response in mice.Moreover, the results of virus neutralization assays suggested that sera from the mice immunized with four proteins elicited high neutralizing activities against the vaccinia virus. Notably, the results of ELISA, ELISPOT, and virus neutralization assays also showed that the CpG7909 adjuvant was more effective in inducing an immune response compared with the aluminum adjuvant. In summary,this study offers valuable insights for further studies of subunit vaccine candidates for the prevention of MPXV and other orthomyxoviruses.