In higher plants, the electron-sink capacity of photorespiration contributes to alleviation of photoinhibition by dissipating excess energy under conditions when photosynthesis is limited. We addressed the question at which point in the evolution of photosynthetic organisms photorespiration began to function as electron sink and replaced the flavodiiron proteins which catalyze the reduction of O2 at photosystem I in cyanobacteria. Algae do not have a higher activity of photorespiration when CO2 assimilation is limited, and it can therefore not act as an electron sink. Using land plants (liverworts, ferns, gymnosperms, and angiosperms) we compared photorespiration activity and estimated the electron flux driven by photorespiration to evaluate its electron-sink capacity at CO2-compensation point. In vivo photorespiration activity was estimated by the simultaneous measurement of O2-exchange rate and chlorophyll fluorescence yield. All C3-plants leaves showed transient O2-uptake after actinic light illumination (post-illumination transient O2-uptake), which reflects photorespiration activity. Post-illumination transient O2-uptake rates increased in the order from liverworts to angiosperms through ferns and gymnosperms. Furthermore, photorespiration-dependent electron flux in photosynthetic linear electron flow was estimated from post-illumination transient O2-uptake rate and compared with the electron flux in photosynthetic linear electron flow in order to evaluate the electron-sink capacity of photorespiration. The electron-sink capacity at the CO2-compensation point also increased in the above order. In gymnosperms photorespiration was determined to be the main electron-sink. C3–C4 intermediate species of Flaveria plants showed photorespiration activity, which intermediate between that of C3- and C4-flaveria species. These results indicate that in the first land plants, liverworts, photorespiration started to function as electron sink. According to our hypothesis, the dramatic increase in partial pressure of O2 in the atmosphere about 0.4 billion years ago made it possible to drive photorespiration with higher activity in liverworts.