In 2022, the largest ever virgin soil outbreak of Japanese encephalitis (JE) occurred in Australia resulting in 45 reported human cases of JE, with seven fatalities. Japanese encephalitis virus (JEV) was detected in 84 piggeries across Australia. In response, states implemented targeted vaccination programs for those individuals at the highest risk of JEV exposure. A mixed methods approach, including geospatial mapping of JEV vaccine distribution in Queensland, a case series of Queensland human cases and interviews with Queensland Health staff, assessed the JEV vaccination response program. Five notified human cases were reviewed, with three having occupational outdoor risk and local travel-related exposure. Vaccine coverage ranged from 0 to 7.4 doses per 100 people after 12 months of the program. The highest uptake was in southern Queensland, where 95% of the state’s commercial pig population is located. The vaccination program was limited by a heavy reliance on general practitioners, vast geographical distribution of eligible populations, difficulties mobilising and engaging eligible cohorts, and suboptimal One Health collaboration. Population and climate factors make it possible for the virus to become endemic. Targeted vaccination programs remain an important strategy to protect people at the highest risk of exposure, however, program improvements are required to optimize vaccine accessibility.