Diatoms are major oceanic primary producers, which sustain marine food webs and contribute to carbon export to the depth. Although the effects of ocean acidification (OA) on the physiological performance of diatoms have been extensively studied, there are a few studies concerning the strain-specific variation in response of diatoms to OA. Here four Phaeodactylum tricornutum strains Pt1, Pt4, Pt8 and PtSCS isolated from different locations worldwide were used to explore the intra-species variation of this model diatom in response to OA at physiological and molecular levels. Our results showed that OA did not significantly influence the photochemical performance and growth rate of P. tricornutum, but significantly down-regulated carbon affinity for carbon fixation in various magnitudes for all these strains. Furthermore P. tricornutum strains showed various expression regulation patterns of some genes involved in biophysical CO2-concentrating mechanisms (CCMs) and C4 metabolism in response to OA. The downregulation of dissolved inorganic carbon (DIC) affinity mainly resulted from the expression regulation of genes involved in biophysical CCMs under OA conditions. Pt4 showed the significant downregulation ofgenes coding bicarbonate transporters SLC4 that was markedly distinct from other strains, which was the main reason of the largest magnitudes of downregulation in DIC affinity induced by elevated CO2, based on the expression patterns of selected key genes involved in carbon assimilation. In addition, our data demonstrated that C4 metabolisms may play important roles in changing mitochondrial CO2, dissipating energy and facilitating lipid biosynthesis under OA conditions.