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Background: Although a vaccination campaign has been conducted since 2004, Japanese encephalitis (JE) is still a public health problem in Guizhou, one of the provinces with the highest incidence of JE in China. The aim of this study was to understand the spatiotemporal distribution of JE and its relationship with environmental factors in Guizhou Province in the post-vaccination era, 2004-2016.Methods: We collected data on human JE cases in Guizhou Province from 2004 to 2016 from the national infectious disease reporting system. A Poisson regression model was used to analyze the relationship between JE occurrence and environmental factors amongst counties.Results: Our results showed that the incidence and mortality of JE decreased after the initiation of vaccination. JE cases were mainly concentrated in preschool and school-age children and the number of cases in children over age 15 years was significantly decreased compared with the previous 10 years; the seasonality of JE before and after the use of vaccines was unchanged. JE incidence was positively associated with cultivated land and negatively associated with gross domestic product (GDP) per capita, vegetation coverage, and developed land. In areas with cultivated land coverage < 25%, vegetation coverage > 55%, and urban area coverage > 25%, the JE risk was lower. The highest JE incidence was among mid-level GDP areas and in moderately urbanized areas.Conclusions: This study assessed the relationship between incidence of JE and environmental factors in Guizhou Province. Our results highlight that the highest risk of JE transmission in the post-vaccination era is in mid-level developed areas. [ABSTRACT FROM AUTHOR]