Recent studies on the diazotrophic cyanobacteria Trichodesmium showed a pronounced CO2 dependency in N2- and C-fixation. However, significant uncertainties remain as to the degree of sensitivity to CO2, modification of these responses by other environmental factors, and the underlying processes. To this end, we investigated carbon acquisition and nitrogen fixation in Trichodesmium IMS101 under different CO2 and light levels. In these acclimations, growth rates, cellular C and N content, and respective isotopic values were measured. In vivo activities of photosynthetic O2 evolution, O2 uptake, CO2 and HCO3- fluxes were obtained using membrane inlet mass spectrometry methods (MIMS). Nitrogen fixation rates were determined using the acetylene reduction assay, and chlorophyll a fluorescence was measured via fluorescence induction and relaxation method (FIRe). Photosynthesis and N2-fixation increased with CO2 and this effect was even more pronounced under high light. Moreover, our data suggest a shift in the distribution of metabolic energy between photosynthesis, carbon acquisition and N2-fixation in Trichodesmium. The observed stimulation in photosynthesis and N2-fixation may enhance the productivity in N-limited oligotrophic regions.