The direct use of industrial flue gas in microalgae production is desired for mitigating CO₂ emissions, but the low pH resulting from the inflow of acidic gases (mainly CO₂, NO_x, and SO_x) imposes detrimental effects on microalgal growth and is considered the main technical challenge for simultaneous biomass production and CO₂ sequestration. In this study, we investigated the adaptive responses of the model marine diatom Phaeodactylum tricornutum to acidic stress at pH 6.0. Gradual changes in the ratio of morphotypes, chlorophyll content, and photosynthetic efficiency were observed as a result of adaptive laboratory evolution (ALE) under constant acidic stress. The evolved strains showed a significant increase in growth rate in acidic conditions after ALE, and phenotypic characterization demonstrated a stable trait of acid tolerance with an average increase in growth by 110.4%, 46.1%, and 27.5% at pH 5.5, 6.0, and 6.5, respectively compared with the parental wild-type strain. Furthermore, RNA sequencing and whole-genome re-sequencing analyses revealed that core pathways, including photosynthesis, pH regulation/ion transport, and carbohydrate and fatty acid metabolism, were upregulated across all three evolved strains, though they exhibited different evolutionary trajectories. This study demonstrated the feasibility of recovering photosynthetic capability after acidic stress in the marine diatom P. tricornutum through ALE and provided molecular data to reveal essential alterations in genetic regulations that could enable cells to tolerate low environmental pH.