Motility plays a critical role in algal survival and reproduction, with implications for aquatic ecosystem stability. However, the effect of elevated CO2 on marine, brackish and freshwater algal motility is unclear. Here we show, using laboratory microscale and field mesoscale experiments, that three typical phytoplankton species had decreased motility with increased CO2. Polar marine Microglena sp., euryhaline Dunaliella salina and freshwater Chlamydomonas reinhardtii were grown under different CO2 concentrations for 5 years. Long-term acclimated Microglena sp. showed substantially decreased photo-responses in all treatments, with a photophobic reaction affecting intracellular calcium concentration. Genes regulating flagellar movement were significantly downregulated (P < 0.05), alongside a significant increase in gene expression for flagellar shedding (P < 0.05). D. salina and C. reinhardtii showed similar results, suggesting that motility changes are common across flagellated species. As the flagella structure and bending mechanism are conserved from unicellular organisms to vertebrates, these results suggest that increasing surface water CO2 concentrations may affect flagellated cells from algae to fish. Algal movement through the water column occurs to maximize photosynthesis and avoid predation. Increased CO2 concentrations are shown, from laboratory and field experiments, to reduce motility in algal species in fresh, brackish and marine systems.