To explore the molecular mechanism of the response of Masson pine (Pinus massoniana), the main coniferous tree in southern China, to high CO2 stress, transcriptome sequencing was carried out to analyze the genome-wide responses of annual seedlings under different durations (0 h, 6 h, 12 h and 24 h) of high CO2 stress. The results showed that a total of 3080/1908, 3110/2115 and 2684/1483 genes were up-/down-regulated after 6 h, 12 h and 24 h of treatment, respectively, compared with control check group (CK, 0 h). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that most of these differentially expressed genes (DEGs) were enriched in energy metabolism, carbohydrate synthesis, cell wall precursor synthesis and hormone regulation pathways. For energy metabolism, the expression of most genes involved in photosynthesis (including the light reaction and Calvin cycle) was generally inhibited, while the expression of genes related glycolysis, the tricarboxylic acid (TCA) cycle and PPP pathway was up-regulated. In addition, the increase in the CO2 concentration induced the up-regulation of gene expression in the sucrose synthesis pathway. Among all starch synthesis genes, GBSS (granule-bound starch synthase) had the highest expression level. On the other hand, during the synthesis of hemicellulose and pectin (cell wall precursor substances), the expression levels of GMD (GDP-mannose 4,6-dehydratase), MGP (Mannose-1-phosphate guanylyl transferase) and RHM (Rhamnose biosynthetic enzyme) were the highest, suggesting that the synthesis of the raw materials hemicellulose and pectin in Masson pine under stress were mainly supplied by GDP-Man, GDP-Fuc and UDP-Rha. Finally, stress inhibited gene expression in the ABA (Abscisic Acid) synthesis pathway and induced gene expression in the GA (Gibberellin), SA (Salicylic acid), BR(Brassinolide) and MeJA (Methyl Jasmonate) pathways. Stomatal switches were regulated by hormonal interactions. This experiment elaborated on the response and molecular mechanism of Masson pine to CO2 stress and aided in screening carbon sequestration genes for the corresponding molecular research of Masson pine in the future.