This study was aimed to determine the changes in CO2 concentration according to the temperatures of daytime and nighttime in the CO2 supplemental greenhouse, and to compare calculated supplementary CO2 concentration during winter and spring cultivation seasons. CO2 concentrations in experimental greenhouses were analyzed by selecting representative days with different average temperatures due to differences in integrated solar radiation at the growth stage of leaf area index (LAI) 2.0 during the winter season of 2022 and 2023 years. The CO2 concentration was 459, 299, 275, and 239 μmol·mol-1, respectively at 1, 2, 3, and 4 p.m. after the CO2 supplementary time (10:00-13:00) under the higher temperature (HT, > 18°C daytime temp. avg. 31.7, 26.8, 23.8, and 22.4°C, respectively), while it was 500, 368, 366, 364 μmol·mol-1, respectively under the lower temperature (LT, < 18°C daytime temp. avg. 22.0, 18.9, 15.0, and 13.7°C, respectively), indicating the CO2 reduction was significantly higher in the HT than that of LT. During the nighttime, the concentration of CO2 gradually increased from 6 p.m. (346 μmol·mol-1) to 3 a.m. (454 μmol·mol-1) in the HT with a rate of 11 μmol·mol-1 per hour (240 tomatoes, leaf area 330m2), while the increase was very lesser under the LT. During the spring season, the CO2 concentration measured just before the start of CO2 fertilization (7:30 a.m.) in the CO2 enrichment greenhouse was 3-4 times higher in the HT (>15°C nighttime temperature avg.) than that of LT (< 15°C nighttime temperature avg.), and the calculated amount of CO2 fertilization on the day was also lower in HT. All the integrated results indicate that CO2 concentrations during the nighttime varies depending on the temperature, and the increased CO2 is a major source of CO2 for photosynthesis after sunrise, and it is necessary to develop a model formula for CO2 supplement considering the nighttime CO2 concentration.