To elucidate a physiological mechanism of heat stress mitigation by exogenous glucose, seedlings of Cucumis sativus cv. Jinchun No. 4 were pretreated with glucose and then exposed to normal (25/18 °C) and elevated (42/38 °C) temperatures. We investigated whether glucose can protect cucumber plantlets and chloroplast ultrastructure from heat and whether this protection is associated with antioxidant enzymes, proline, and soluble sugars. Heat inhibited plant growth, disorganized membranes of 86.33 % of chloroplasts, and elevated the content of malondialdehyde (MDA), superoxide radical (O₂ ^·−), and hydrogen peroxide (H₂O₂). An optimum concentration of glucose was 30 mM as it significantly alleviated plant growth inhibition and obviously reduced the content of MDA, O₂ ^·−, and H₂O₂ under the heat stress. The pretreatment with 30 mM glucose mitigated heat-induced damage of chloroplast ultrastructure and changes in leaf morphology more than 30 mM mannitol suggesting that glucose did not act only as osmolyte. Moreover, the glucose pretreatment increased activities of some antioxidant enzymes and enhanced the content of proline and soluble sugars under the heat stress, as well as the transcriptions of Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, catalase, and glutathione reductase genes. We conclude that the pretreatment with 30 mM glucose protected chloroplast ultrastructure and enhanced heat tolerance of the seedlings by the increased activites of antioxidants and the content of proline and soluble sugars, and repressed accumulation of reactive oxygen species.