ABSTRACT: Tea (Camellia sinensis (L.) O. Kuntze), one of the main crops in China, is high in various bioactive compounds including flavonoids, catechins, caffeine, theanine, and other amino acids. C. sinensis is also known as an accumulator of fluoride (F), and the bioactive compounds are affected by F, however, the mechanism remains unclear. Here, the effects of F treatment on the accumulation of F and major bioactive compounds and gene expression were investigated, revealing the molecular mechanisms affecting the accumulation of bioactive compounds by F treatment. The results showed that F accumulation in tea leaves gradually increased under exogenous F treatments. Similarly, the flavonoid content also increased in the F treatment. In contrast, the polyphenol content, free amino acids, and the total catechins decreased significantly. Special amino acids, such as sulfur-containing amino acids and proline, had the opposite trend of free amino acids. Caffeine was obviously induced by exogenous F, while the theanine content peaked after two day-treatment. These results suggest that the F accumulation and content of bioactive compounds were dramatically affected by F treatment. Furthermore, differentially expressed genes (DEGs) related to the metabolism of main bioactive compounds and amino acids, especially the pivotal regulatory genes of catechins, caffeine, and theanine biosynthesis pathways, were identified and analyzed using high-throughput Illumina RNA-Seq technology and qRT-PCR. The expression of pivotal regulatory genes is consistent with the changes of the main bioactive compounds in C. sinensis leaves, indicating a complicated molecular mechanism for the above findings. Overall, these data provide a reference for exploring the possible molecular mechanism of the accumulation of major bioactive components such as flavonoid, catechins, caffeine, theanine and other amino acids in tea leaves in response to fluoride treatment.