Differential responses of diatoms, an important group of marine primary producers to ocean acidification, have been well documented. However, studies so far are based on limited representative strains from key species. Investigation of strain level responses will help us better understand the contrasting discrepancy in diatom responses to ocean acidification. Here, we selected four strains of the model diatom Phaeodactylum tricornutum isolated from different regions of the global ocean, representing all genotypes based on internal transcribed spacer 2 sequences, and investigated strain‐specific responses to ocean acidification. In response to ocean acidification, changes in carbon metabolism varied among strains, although no significant effects of ocean acidification on growth rates or pigments were observed in any strains. The expression of genes encoding plasma membrane bicarbonate transporters was downregulated in strain Pt4, reflecting a potential decrease in active HCO3− uptake, which was not observed in the other strains. Reduction of CO2 concentrating mechanism efficiency was also indicated by the regulated expression of genes encoding carbonic anhydrases that catalyze the interconversion of HCO3− and CO2 in the pyrenoids and pyrenoid‐penetrating thylakoid, which exhibited different patterns among the strains. Under ocean acidification conditions, C4‐like metabolism appeared to redistribute carbon flux to gluconeogenesis in strain Pt1, and lipid synthesis in strains Pt8 and Pt11, rather than participating in net photosynthetic carbon fixation. These variations were incompletely correlated with phylogenetic relationship in different strains, implying that the habitat‐adapted imprints of the different strains could also be responsible for their differential responses to ocean acidification.