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Despite being a key enzyme of Cavin cycle, transketolase (TK) is believed to be related to abiotic resistance in higher plants. However, how TK affects chilling tolerance still remains largely unknown. Here, we describe the effect of overexpression of the Cucumis sativa TK gene (CsTK) on growth, photosynthesis, ROS metabolism and cell ultrastructure under chilling stress. Low temperature led to a decrease of the photosynthetic rate (Pn), the stomatal conductance (Gs), the actual photochemical efficiency (ΦPSII) and the sucrose content, whereas there was an increase of the intercellular CO 2 concentration (Ci) and MDA content. These changes were alleviated in the CsTK plants after 5 days of chilling stress, however, inhibition of CsTK showed the opposite results. Furthermore, transgenic plants with overexpression of CsTK showed higher increase in leaf area and dry matter, higher activity of the enzymes and higher increase in the contents of metabolism substance involved in Calvin cycle and reactive oxygen scavenging system as well as lower • OH and H 2 O 2 content, superoxide anion production rate compared with the control cucumber plants under chilling stress. At the end of the chilling stress, compared to wild-type (WT) which exhibited dramatically destroyed cell ultrastructure, expanded chloroplast, broken cell and chloroplast membranes as well as the disappeared grana lamella, the CsTK sense plants showed a more complete cell ultrastructure, whereas, the damage of the cell ultrastructure was aggravated in CsTK antisense plants. Taken together, these results imply that CsTK promoted chilling tolerance in cucumber plants mainly through increasing the capacity to assimilate carbon, alleviating oxidative damage and stabilizing cell structure.