With increasing H5 avian influenza cases reported globally and the potential for pandemic emergence, induction of cross-reactive antibody responses may represent an important attribute of an effective vaccine. This phase 2 extension study evaluated immunogenicity and safety of MF59-adjuvanted, cell culture-derived H5N6 vaccine (aH5N6c) in adults primed with MF59-adjuvanted, cell culture-derived H5N1 vaccine (aH5N1c) and in unprimed adults. Adults previously primed with two doses of aH5N1c in the parent study V89_18 were randomized to receive two aH5N6c doses (Group 1) or one aH5N6c and one placebo (Group 2) 3?weeks apart. Unprimed adults received two aH5N6c doses (Group 3). Immunogenicity was assessed by hemagglutination inhibition (HI) and microneutralization (MN) assays against the priming (H5N1) and booster (H5N6) strains on Days 1, 8, 22, 43, and 202. Among 258 exposed participants, primed subjects (Groups 1 and 2) showed higher HI geometric mean titers against both strains than unprimed (Group 3) subjects, with MN responses similarly enhanced. Heterologous H5N1 responses were robust in primed subjects (Day 43 HI GMTs: 333–343; seroconversion rates >89%) but minimal in unprimed subjects, with responses persisting to Day 202. Solicited adverse events were mild or moderate, comparable between groups, and consistent with other MF59-adjuvanted pandemic vaccines; no vaccine-related serious adverse events occurred. Heterologous H5N6 booster vaccination in H5N1-primed adults elicited strong cross-reactive immunity against the priming strain, demonstrating long-lasting immune memory for at least 6 y and supporting heterologous prime-boost strategies for pandemic preparedness against emerging H5 outbreaks.