One of the great challenges in biogeochemical research over the past half a century has been to quantify and understand the mechanisms underlying stable carbon isotope fractionation (εp) in phytoplankton in response to changing CO2 concentrations. Partly, this interest is grounded in the use of fossil photosynthetic organism remains as a proxy for past atmospheric CO2 levels. Phytoplankton organic carbon is depleted in 13C compared to its source because of kinetic fractionation by the enzyme RubisCO during photosynthetic carbon fixation, as well as through physiological pathways upstream of RubisCO. Moreover, other factors such as nutrient limitation, variations in light regime as well as phytoplankton culturing systems and inorganic carbon manipulation approaches may confound the influence of aquatic CO2 concentration ([CO2]) on εp. Here, based on experimental data compiled from the literature, we assess which underlying physiological processes cause the observed differences in εp for various phytoplankton groups in response to C-demand/C-supply (i.e., POC production/[CO2]) and test potential confounding factors. Culturing approaches and methods of carbonate chemistry manipulation were found to best explain the differences in εp between studies, although daylength was an important predictor for εp in haptophytes. Extrapolating results from culturing experiments to natural environments and for proxy applications therefore requires caution, and it should be carefully considered whether culture methods and experimental conditions are representative of natural environments.