The regulation of carbon partitioning in diatoms is key to understanding mechanisms related to their high productivity, and the mechanisms controlling carbon flux towards different metabolic fates are currently not well understood. A unique variant of 6-phosphofructo-2-kinase/fructose 2,6 bisphosphatase (PFK2/F2BP), a regulator of glycolytic flux previously uncharacterized in single-cell photosynthetic eukaryotes, was identified in diatom genomes and overexpressed in Thalassiosira pseudonana. Overexpression resulted in increased activity of the glycolytic rate-determining enzyme phosphofructokinase (PFK) and altered carbon partitioning among intracellular carbohydrate, lipid, and protein levels. Higher PFK activity in transformant lines correlated with reduced growth rate and an extension of the G1 phase of the cell cycle, demonstrating a link between carbon metabolism and the control of cell cycle processes. While the relationship between growth and carbon flux under environmental stress (such as nutrient limitation) is well documented in diatoms, our data demonstrate that manipulation of carbon metabolism, independent of an environmental trigger (such as nutrient limitation), is sufficient to delay cell cycle progression. The inverse relationship between growth and PFK activity (as an indicator of glycolytic flux) differs from those described for other unicellular eukaryotes and supports an alternate organization and regulation of central carbon metabolism in diatoms, including a prominent regulatory role for PFK2/F2BP.