The synchronous origin of agriculture in at least four independent climatic regions at the end of the last glacial period (c10 kyr bp) points to a global limitation for crop domestication. One hypothesis proposes that a rapid carbon dioxide (CO2) increase from 18 Pa to ∼27 Pa during deglaciation caused significant increases in the growth rates of wild crop progenitors, thereby removing a productivity barrier to their successful domestication. However, early C4 crops present a challenge to this hypothesis, because they were among the first domesticates, but have a carbon-concentrating mechanism that offsets the limitation of photosynthesis by CO2. We investigated the CO2-limitation hypothesis using the wild progenitors of five C4 founder crops from four independent centres of domestication. Plants were grown in controlled environment chambers at glacial (18 Pa), postglacial (28 Pa) and current ambient (38 Pa) CO2 levels, and photosynthesis, transpiration and biomass were measured. An increase in CO2 from glacial to postglacial levels caused a significant gain in vegetative biomass of up to 40%, but the equivalent rise in CO2 from postglacial to modern levels generally had no effect on biomass. Investigation into the underlying mechanisms showed C4 photosynthesis to be limited more by glacial than postglacial CO2 levels, matching theoretical expectations. Moreover, the increase in CO2 from glacial to postglacial levels caused a reduction in the transpiration rate via decreases in stomatal conductance of ∼35%. In combination, these physiological changes conferred a large improvement in water-use efficiency at the postglacial CO2 partial pressure compared with the glacial level. Our data, therefore, provide experimental support for the CO2-limitation hypothesis, suggesting that these key physiological changes could have greatly enhanced the productivity of wild crop progenitors after deglaciation.