Acidification of seawater caused by anthropogenic carbon dioxide (CO2) is anticipated to influence the growth of dinitrogen (N-2)-fixing phytoplankton, which contribute a large fraction of primary production in the tropical and subtropical ocean. We found that growth and N-2-fixation of the ubiquitous cyanobacterium Trichodesmium decreased under acidified conditions, notwithstanding a beneficial effect of high CO2. Acidification resulted in low cytosolic pH and reduced N-2-fixation rates despite elevated nitrogenase concentrations. Low cytosolic pH required increased proton pumping across the thylakoid membrane and elevated adenosine triphosphate production. These requirements were not satisfied under field or experimental iron-limiting conditions, which greatly amplified the negative effect of acidification.