Abstract: In this study, Isoetes orientalis Liu Hong was transplanted at different altitudes, and gene expression analysis was performed based on transcriptome sequencing to explore its adaptation mechanism in aquatic and terrestrial environments at different altitudes. Results showed that, compared with low-altitude transplants, high-altitude transplants exhibited more differentially expressed genes significantly enriched in inositol phosphate metabolism, photosynthesis, antenna protein, flavonoid, and phenylpropanoid biosynthesis pathways. In particular, many specifically expressed genes were identified in plants transplanted to Tibet, most of which were annotated as ribosomal proteins, DNA damage repair enzymes, and enzymes related to unsaturated fatty acid biosynthesis. This gene expression pattern may be related to plant adaptation to high-altitude environments with low oxygen, strong radiation, and marked temperature changes. Results also showed that I. orientalis grown at low altitude long-term could survive in high-altitude environments, and there were certain rules in gene expression patterns. This study provides a reference for exploring the adaptation mechanisms of Isoetes plants to aquatic and terrestrial environments at different altitudes.