Abstract： Objective:To evaluate the vaccine effectiveness (VE) of enterovirus A71 (EV-A71) inactivated vaccine against severe cases associated with EV-A71.Methods:A test-negative design (TND) case-control study was carried out from January 1, 2019 to December 31, 2021 in Hunan, Henan and Yunnan province to assess the VE of EV-A71 vaccine in preventing severe HFMD.Results:In total, 896 severe HFMD patients aged 6 to 71 months were included in the study, with a positive enterovirrus test rate of 83.3%. For these patients, 248 (33.2%) were CVA6-positive, 142 (19.0%) were CVA16-positive, 62 (8.3%) were CVA10-positive, 52 cases (7.0%) were EV-A71 positive, and 242 cases (32.4%) were not typed. Of the EV-A71-positive cases, none had received EV-A71 vaccine, the EV-71 vaccine against severe cases of EV-A71-associated HFMD was 100%, and did not cross-protect against CVA16, CVA10, or other enterovirus infections.Conclusions:The EV-A71 vaccine demonstrated high level VE against severe cases of EV-A71-associated HFMD. Presently, the dominant serotype of enteroviruses in severe cases of HFMD had changed. Under the circumstances, multivalent vaccine should be developed for the prevention of other enteroviral infections, especially for serotypes such as CVA6 and CVA16.