Encephalomyocarditis virus (EMCV) is a pandemic virus that has caused mortality in numerous captive wildlife species worldwide. An experimental killed vaccine was created from two EMCV isolates associated with zoo animal mortality in the southern United States. The vaccine was tested for safety and efficacy in eleven llamas (Llama glama). All animals received an initial vaccination and a second booster vaccination 4 wk later. Serum antibody responses were monitored at initial vaccination and at 4 wks, 8 wk, 6 mo, and 12 mo postvaccination. Eight of the 11 llamas vaccinated experienced at least a 4-fold increase in serum antibody titers to EMCV. Antibody titers of those eight animals remained elevated above prevaccination levels when measured at 12 mo. The experimental killed EMCV vaccine tested may be a useful tool to prevent EMCV infection in llamas when given in 2 doses 4 wk apart, and then revaccinated or with antibody levels monitored annually thereafter.