Raising temperature-induced heat stress under climate warming scenarios has become a predominant threat to crop growth and productivity. As a pleiotropic signaling molecule, melatonin offers an innovative solution for enhancing plant thermotolerance, although its mechanisms, particularly regarding leaf photosynthesis, remain insufficiently understood. This study employed proteomic and physiological analyses to reveal the potential benefits of endogenous melatonin in alleviating heat stress-induced damage to the photosynthetic performance of Nicotiana tabacum plants. Foliar application of melatonin at 50 μM effectively ameliorated heat stress induced-photoinhibition by preventing pigment degradation, enhancing Rubisco and FBPase activities, stimulating RuBP carboxylation and regeneration, and improving light energy transfer and utilization.The changes resulted in increased light-saturated photosynthesis rate and photochemical efficiency. Melatonin application also elevated starch and soluble sugar contents by stimulating photosynthetic carbon assimilation and suppressing dark respiration, thereby counteracting the harmful impact of heat stress. Proteomic analysis revealed that melatonin significantly upregulated the expression of two key enzymes (glutamyl-tRNA reductase and monomethyl ester aerobic oxidative cyclase) involved in the chlorophyll biosynthetic pathway, enhanced the expression of three proteins (PSII cytochrome b559, protein H, and 10 kDa polypeptide) related to the PSII photochemical reaction, stimulated the expression of fructose-1,6-bisphosphatase linked to the Calvin cycle, and increased the expression of granule-bound starch synthase related to carbohydrate metabolism, thereby positively mediating the photodamage induced by heat stress to plant photosynthetic performance. These results highlight the potential of endogenous melatonin application as an effective approach for boosting crop photosynthetic performance and thermotolerance to global warming.