Autotrophy is the basis for complex life on Earth. Its physiological limits are largely constrained by rubisco - the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 prokaryotic form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco’s carboxylation velocity. We show that phototrophy and the presence of a carboxysome CO2concentrating mechanism correlate with faster rubiscos (median rate difference of 3-5 fold). In contrast to previous assumptions, rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s-1median versus