Summary: Investigating immune memory to vaccinia virus and pre-existing immunity to mpox virus (MPXV) among the population is crucial for the global response to this ongoing mpox epidemic. Blood was sampled from vaccinees inoculated with vaccinia virus Tiantan (VTT) strain born before 1981 and unvaccinated control subjects born since 1982. After at least 40 years of the inoculation, 60% or 5% VTT vaccinees possess neutralizing antibodies (NAbs) to VTT or MPXV, with at least 50% having T cell memory to VTT protein antigens. Notably, 46.7% vaccinees show pre-existing T cell responses to MPXV. Broad pre-existing CD8+ T cell reactivities to MPXV are detected not only against conserved epitopes but also against variant epitopes between VTT and MPXV. Persistent NAbs and T cell memory to VTT among vaccinees, along with pre-existing T cells to MPXV among both vaccinees and the unvaccinated population, indicate a particular immune barrier to mpox.