CO2 concentration and temperature for growth of photosynthetic organisms are two important factors to ensure better photosynthetic performance. In this study, we investigated the effects of CO2 concentration and temperature on the photosynthetic performance in a marine centric diatom Chaetoceros gracilis. Cells were grown under four different conditions, namely, at 25 °C with air bubbling, at 25 °C with a supplementation of 3% CO2, at 30 °C with air bubbling, and at 30 °C with the CO2 supplementation. It was found that the growth rate of cells at 30 °C with the CO2 supplementation is faster than those at other three conditions. The pigment compositions of cells grown under the different conditions are altered, and fluorescence spectra measured at 77 K also showed different peak positions. A novel fucoxanthin chlorophyll a/c-binding protein complex is observed in the cells grown at 30 °C with the CO2 supplementation but not in the other three types of cells. Since oxygen-evolving activities of the four types of cells are almost unchanged, it is suggested that the CO2 supplementation and growth temperature are involved in the regulation of photosynthetic light-harvesting apparatus in C. gracilis at different degrees. Based on these observations, we discuss the favorable growth conditions for C. gracilis.