Japanese encephalitis (JE) is a mosquito-borne viral disease, which is the most serious viral encephalitis in China and other countries of the Asia-Pacific region. Since 2005, the epidemic patterns of JE have changed dramatically in China because of the vaccination of children younger than 15 years old, and JE is expanding geographically along with global warming. This retrospective epidemiological study analyzed dynamic environmental factors and the spatio-temporal distribution of human cases of JE in Shaanxi Province-one of the most severely affected areas of China-from 2005 to 2018. The results demonstrated that the high-risk population changed rapidly as the annual rate of JE cases increased by more than 40% in the age group >60 years during the study period, and endemic areas expanded northward in Shaanxi. Hotspot analysis detected four hotspots accounting for 52.38% the total cases, and the panel negative binomial regression model revealed that the spatio-temporal distribution of JE was significantly affected by temperature, relative humidity, wind velocity, El Niño-Southern Oscillation, coniferous forest coverage, and urban areas. These findings can provide useful information for improving current strategies and measures to reduce disease incidence.