Carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are critical enzymes involved in the CO2-concentrating mechanism (CCM) and carbon fixation in phytoplankton. These enzymes play key roles in photosynthetic carbon acquisition and fixation in diatoms. However, the potential synergistic interactions of the said enzymes remain little understood. In the present study, effects of CA inhibitors on the growth, photosynthetic rate, and Rubisco activities in the diatom Phaeodactylum tricornutum were investigated. Our results showed that the growth and photosynthetic O2 evolution rate of P. tricornutum were significantly inhibited by the intracellular CA (iCA) inhibitor ethoxzolamide (EZ), but not by extracellular CA (eCA) inhibitor acetazolamide (AZ) in the long term. This suggests that the eCA was not necessarily essential for growth and photosynthesis, but rather it was the iCA activity that was more crucial in P. tricornutum. We also found that there is a significant positive correlation between Rubisco and CA activities, suggesting that the two carbon acquisition and fixation components of photosynthesis may have undergone co-evolution to optimize fundamental trade-offs. These results further indicate that the roles of eCA and iCA may also differ among diatom species or strains, and that their functions are tightly correlated with other carbon fixation components.