Diatoms are responsible for a large proportion of global carbon fixation, with the possibility that they may fix more carbon under future levels of high CO2. To determine how increased CO2 concentrations impact the physiology of the diatom Thalassiosira pseudonana Hasle et Heimdal, nitrate-limited chemostats were used to acclimate cells to a recent past (333 ± 6 μatm) and two projected future concentrations (476 ± 18 μatm, 816 ± 35 μatm) of CO2. Samples were harvested under steady-state growth conditions after either an abrupt (15–16 generations) or a longer acclimation process (33–57 generations) to increased CO2 concentrations. The use of un-bubbled chemostat cultures allowed us to calculate the uptake ratio of dissolved inorganic carbon relative to dissolved inorganic nitrogen (DIC:DIN), which was strongly correlated with fCO2 in the shorter acclimations but not in the longer acclimations. Both CO2 treatment and acclimation time significantly affected the DIC:DIN uptake ratio. Chlorophyll a per cell decreased under elevated CO2 and the rates of photosynthesis and respiration decreased significantly under higher levels of CO2. These results suggest that T. pseudonana shifts carbon and energy fluxes in response to high CO2 and that acclimation time has a strong effect on the physiological response.