Current knowledge about what the protective roles of elevated CO2 concentration (eCO2) on chlorophyll fluorescence, PSII photochemistry and antioxidant capacity of Arabidopsis with heat stress remains insufficient. Besides, there is a lack of information on the tolerance mechanism of plants against stress in the presence or absence of sirtuin genes. This study aimed to investigate how eCO2 (800 ppm) mitigated heat stress (36 °C)-dependent oxidative damages through analyzing growth, water content, gas exchange, chlorophyll fluorescence, electron flux efficiency in PSII, antioxidant capacity and lipid peroxidation in Arabidopsis thaliana cv. Columbia-0 (Col-0) and its sirtuin-deficient mutant (srt2) plants. The focus was on investigating the function of the SRT2 gene, particularly in comparison with wild-type plants. Therefore, the sirtuin inhibitor (sirtinol, 1 µM S) was applied to Col-0 plants and the effects of ambient (400 ppm) and eCO2 heat (H) under stress were also examined. The reductions in growth and water content of heat stress-treated Col-0 and srt2 mutants were eliminated by eCO2 exposure. The elevated CO2 was removed the reductions on chlorophyll fluorescence, carbon assimilation rate and carboxylation efficiency dependent by H stress in Col-0 plants, but not in srt2 mutants. S supplementation to Col-0 plants produced effects similar to those observed under stress across all measured parameters. While the structural indicators and electron fluxes of PSII and performance indices were disrupted by H stress, eCO2 provided the positive effects on the photochemistry of PSII in the wild type of Arabidopsis. Interestingly, the eCO2-triggered alleviation against stress in srt2 mutants was not maintain. The induced (SOD and GR) and reduced activities (POX, APX, AsA/DHA and GSH redox state) of antioxidant systems point to increased H2O2 accumulation in both Col-0 (123%) and srt2 mutant (40%). Both of the Arabidopsis plants experienced oxidative stress as proved by the high levels of lipid peroxidation. eCO2 under H stress resulted in remarkable decline in H2O2 accumulation, which contributed to POX and AsA regeneration in Col-0 and POX, AsA and GSH redox status in srt2. Sirtinol application to Col-0 plants also eliminated eCO2-mediated protection on TBARS content. This study provides the first evidence of SRT2 involvement in CO₂-mediated heat stress tolerance.