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Isoprenoids are a functionally and structurally diverse class of natural organic chemicals. The universal precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate are synthesized through the mevalonate and 2C-methyl-D-erythritol 4-phosphate (MEP) pathways, respectively. Many isoprenoids produced through the MEP pathway play an important role in plant acclimation to different light environments. Eupatorium adenophorum, an invasive weed in China, presents a remarkable capacity to acclimate to various light environments, which constitutes its solid foundation of being a successful invasive species. Thus we aimed at gaining a deeper insight into the regulation of MEP pathway in E. adenophorum to further understand the invasive mechanism. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF or MCS) is an essential enzyme in the MEP pathway. In this paper, a novel IspF gene was cloned and characterized from E. adenophorum. Tissue-speciifc expression assays revealed a higher expression of EaIspF1 in leaves than in stems and roots. The expression of EaIspF1 was responsive to different light conditions. Some up-regulation of EaIspF1 expression was also found after the treatments with signal compounds and after wounding stress. Interestingly, the over-expression of EaIspF1 in Arabidopsis led to increase carotenoids contents, resulting in an enhanced tolerance to high light. Taken together, these results indicate that the EaIspF1-derived enzyme participates in isoprenoid metabolism and among others, the expression of this gene in E. adenophorum is involved in the regulation of plastidial isoprenoids, which play an important role in acclimation to various light environments.