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Background Animal control measures in Latin America have decreased the incidence of urban human rabies transmitted by dogs and cats; currently most cases of human rabies are transmitted by bats. In 2004–2005, rabies outbreaks in populations living in rural Brazil prompted widespread vaccination of exposed and at-risk populations. More than 3,500 inhabitants of Augusto Correa (Pará State) received either post-exposure (PEP) or pre-exposure (PrEP) prophylaxis. This study evaluated the persistence of rabies virus-neutralizing antibodies (RVNA) annually for 4 years post-vaccination. The aim was to evaluate the impact of rabies PrEP and PEP in a population at risk living in a rural setting to help improve management of vampire bat exposure and provide additional data on the need for booster vaccination against rabies. Methodology/Principal Findings This prospective study was conducted in 2007 through 2009 in a population previously vaccinated in 2005; study participants were followed-up annually. An RVNA titer >0.5 International Units (IU)/mL was chosen as the threshold of seroconversion. Participants with titers ≤0.5 IU/mL or Equivalent Units (EU)/mL at enrollment or at subsequent annual visits received booster doses of purified Vero cell rabies vaccine (PVRV). Adherence of the participants from this Amazonian community to the study protocol was excellent, with 428 of the 509 (84%) who attended the first interview in 2007 returning for the final visit in 2009. The long-term RVNA persistence was good, with 85–88.0% of the non-boosted participants evaluated at each yearly follow-up visit remaining seroconverted. Similar RVNA persistence profiles were observed in participants originally given PEP or PrEP in 2005, and the GMT of the study population remained >1 IU/mL 4 years after vaccination. At the end of the study, 51 subjects (11.9% of the interviewed population) had received at least one dose of booster since their vaccination in 2005. Conclusions/Significance This study and the events preceding it underscore the need for the health authorities in rabies enzootic countries to decide on the best strategies and timing for the introduction of routine rabies PrEP vaccination in affected areas., Author Summary Animal control measures have decreased the incidence of human rabies in urban regions of Latin America. Currently, most cases of human rabies occur in rural areas and are transmitted by bats. In 2004–2005, rabies outbreaks affecting populations living in remote areas of Brazil prompted the widespread vaccination of exposed and at-risk populations. We evaluated the persistence of the humoral immune response for 4 years after vaccination in a rural population at daily risk of rabies exposure. Our aim was to evaluate the impact of vaccination in a rural setting to help improve management of vampire bat exposure. The participation of this Amazonian community was excellent, with 428 of the 509 (84%) who attended the first interview in 2007 returning for the final visit in 2009. The long-term RVNA persistence was good, with 85–88% of the participants evaluated at each yearly follow-up visit remaining seroconverted. Similar neutralizing antibody persistence levels were observed in participants originally given post-exposure or pre-exposure prophylaxis in 2005. This study and the events preceding it underscore the need for the health authorities in rabies enzootic countries to decide on the best strategies and timing for the introduction of routine rabies PrEP vaccination in affected areas.