Abstract Atmospheric CO 2 level is one of the most important factors which affect plant growth and crop production. Although many crucial genes and pathways have been identified in response to atmospheric CO 2 changes, the integrated and precise mechanisms of plant CO 2 response are not well understood. Alternative splicing (AS) is an important gene regulation process that affects many biological processes in plants. However, the AS pattern changes in plants in response to elevated CO 2 levels have not yet been investigated. Here, we used RNA-Seq data of Arabidopsis thaliana grown under different CO 2 concentration to analyze the global changes in AS. We found that AS increased with the rise in CO 2 concentration. Additionally, we identified 345 differentially expressed (DE) genes and 251 differentially alternative splicing (DAS) genes under the elevated CO 2 condition. Moreover, the results showed that the expression of most of the DAS genes did not change significantly, indicating that AS can serve as an independent mechanism for gene regulation in response to elevated CO 2. Furthermore, our analysis of function categories revealed that the DAS genes were associated mainly with the stimulus response. Overall, this the first study to explore the changes of AS in plants in response to elevated CO 2. Highlights • Comparative transcriptomics analysis of Arabidopsis thaliana grown different CO 2 concentration were performed. • Elevated CO 2 concentration can cause alternative splicing change in Arabidopsis thaliana. • Alternative splicing served as an independent mechanism for gene regulation in response to elevated CO 2. [ABSTRACT FROM AUTHOR]