This study aims to elucidate the molecular mechanism for the transient increase in the O2-uptake rate in tobacco (Nicotiana tabacum cv Xanthi) leaves after turning off actinic lights (ALs). The photosynthetic O2 evolution rate reaches a maximum shortly after the onset of illumination with ALs and then decreases to zero in atmospheric CO2/O2 conditions. After turning off the ALs, tobacco leaves show a transient increase in the O2-uptake rate, the post-illumination transient O2-uptake, and thereafter, the O2-uptake rate decreases to the level of the dark-respiration rate. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], maintained a steady-state value distinct from the photosynthetic O2-evolution rate. In high-[CO2] conditions, the photosynthetic O2-evolution rate and Y(II) showed a parallel behavior, and the post-illumination transient O2-uptake was suppressed. On the other hand, in maize leaves (a C4 plant), even in atmospheric CO2/O2 conditions, Y(II) paralleled the photosynthetic O2-evolution rate and the post-illumination transient O2-uptake was suppressed. Hypothesizing that the post-illumination transient O2-uptake is driven by C3 plant photorespiration in tobacco leaves, we calculated both the ribulose 1,5-bisphosphate carboxylase- and oxygenase-rates (Vc and Vo) from photosynthetic O2-evolution and the post-illumination transient O2-uptake rates. These values corresponded to those estimated from simultaneous chlorophyll fluorescence/O2-exchange analysis. Furthermore, the H+-consumption rate for ATP synthesis in both photosynthesis and photorespiration, calculated from both Vc and Vo that were estimated from chlorophyll fluorescence/CO2-exchange analysis, showed a positive linear relationship with the dissipation rate of the electrochromic shift signal. Thus, these findings support our hypothesis.