Iron is limiting phytoplankton productivity in large parts of today's oceans, the so-called HNLC (high nutrient low chlorophyll) areas. It is a key component in photosynthesis during which inorganic carbon fixation in most phytoplankton species is sustained by so-called carbon concentrating mechanisms (CCMs). Here we investigate CCM regulation in the coccolithophore Emiliania huxleyi in response to varying degrees of iron limitation by means of membrane-inlet mass spectrometry. Compared to iron replete conditions rates of both active CO2 and HCO-3 uptake were markedly reduced under iron limitation leading to significantly diminished growth rates. Moreover, there was a concomitant decrease in CCM efficiency, reflected in an increased CO2 loss from the cell in relation to carbon fixation. Under such conditions higher values for carbon isotope fractionation (∈P) would be expected. However, direct measurements of ∈P showed that carbon isotope fractionation was insensitive to changes in growth rates and CCM activity. This can be explained by concomitant changes in internal DIC fluxes in and out of the chloroplast as demonstrated with a simple cell model comprising two compartments. Thus, carbon isotope fractionation reflects the ability of phytoplankton to actively control their inorganic carbon acquisition depending on environmental conditions. The insensitivity of carbon isotope fractionation to changes in the availability of iron could be of interest for paleoreconstructions in the HNLC areas of today's oceans.