Background: Limited data exist on antibody responses to mixed vaccination strategies that involve inactivated coronavirus disease 2019 (COVID-19) vaccines, particularly in the context of emerging variants. Methods: We conducted an open-label trial of a third vaccine dose of a messenger RNA (mRNA) vaccine (BNT162b2, Fosun Pharma/BioNTech) in adults aged ≥30 years who had previously received 2 doses of inactivated COVID-19 vaccine. We collected blood samples before administering the third dose and 28 days later and tested for antibodies to the ancestral virus using a binding assay (enzyme-linked immunosorbent assay [ELISA]), a surrogate virus neutralization test (sVNT), and a live virus plaque reduction neutralization test (PRNT). We also tested for antibodies against the Omicron variant using live-virus PRNT. Results: In 315 participants, a third dose of BNT162b2 substantially increased antibody titers on each assay. Mean ELISA levels increased from an optical density of 0.3 to 2.2 (P <. 001), and mean sVNT levels increased from an inhibition of 17% to 96% (P <. 001). In a random subset of 20 participants, the geometric mean PRNT50 titers rose substantially, by 45-fold from day 0 to day 28 against the ancestral virus (P <. 001) and by 11-fold against the Omicron variant (P <. 001). In daily monitoring, post-vaccination reactions subsided within 7 days for more than 99% of participants. Conclusions: A third dose of COVID-19 vaccine with an mRNA vaccine substantially improved antibody levels against the ancestral virus and the Omicron variant with a well-tolerated safety profile in adults who had received 2 doses of inactivated vaccine 6 months earlier.