OBJECTIVES: The data on immune responses of individuals undergoing modified vaccinia Ankara-Bavarian Nordic (MVA-BN) vaccination are scarce. We aimed to compare Mpox virus-specific antibody and neutralizing antibody responses among people with and those without HIV receiving MVA-BN vaccines. METHODS: This prospective study enrolled participants undergoing two-dose MVA-BN vaccination to investigate seroresponses after vaccination. Blood samples were collected before and after each dose of vaccination for determinations of anti-A29 and anti-H3 IgG. Neutralization tests were conducted for samples tested positive for both anti-A29 and anti-H3 IgG. RESULTS: Overall, 441 participants undergoing two-dose MVA-BN vaccination were enrolled. Seroconversion for anti-A29 and anti-H3 IgG, respectively, after the second dose of vaccination was 18.2% and 61.2%, 10.9% and 65.0%, and 51.6% and 90.6% among people with HIV, people without HIV, and those who had had smallpox vaccination previously, respectively. About 20% of the participants with seroconversion lost seroresponses after a 7-month period of observation. None of the serum samples from vaccinated participants demonstrated neutralizing ability. CONCLUSIONS: Participants with previous smallpox vaccination had higher and more sustained antibody responses after receiving two doses of MVA-BN vaccines than those who had not undergone smallpox vaccination. More studies are warranted to assess the seroresponses to booster MVA-BN vaccination for vaccine nonresponders or those having lost seroresponses.