Photorespiration has been indicated as an important mechanism for maintaining CO2 assimilation and alleviating photodamage under conditions of high light and low CO2. We tested the hypothesis that plants grown under a high temperature had greater electron flow for photorespiration compared with those grown under a relative low temperature. Responses of photosynthetic electron flow and CO2 assimilation to incident light intensity and intercellular CO2 concentration were examined in leaves of tobacco cultivar ‘k326’. Plants were cultivated at three sites with different ambient temperatures (Zhengzhou, Zunyi and Jiangchuan). Under high light, plants grown in Zhengzhou (with the highest growth temperature in the three sites) showed higher effective quantum yield of photosystem II and total electron flow through photosystem II than that in Zunyi and Jiangchuan. However, regardless of light intensity and intercellular CO2 status, there were no significant differences among sites in the photosynthetic CO2 assimilation rate or electron flow devoted to the carboxylation of ribulose-1,5-bisphosphate (RuBP). As a result, plants grown at high temperature showed higher electron flow devoted to oxygenation of RuBP than plants grown at low temperature. These results suggested that enhancement of electron flow for photorespiration is an important strategy in tobacco for acclimating to high growth temperature.