Sudden changes in environmental conditions exerted on algal cells can greatly affect cell growth and development. To survive these situations, cells need to acclimate and adapt to a new environment. Acclimation is the enhanced ability of cells to survive a more severe dose of stress if the cells have experienced a milder stress beforehand. Heat stress induces production of reactive oxygen species. In plants and algae, reactive oxygen species (ROS) play an integral role as signalling molecules in the regulation of numerous biological processes such as growth development, and responses to biotic and abiotic stimuli in cell. To investigate the effect of mild heat treatment and heat stress on the physiology of C. reinhardtii cells especially on anti-oxidant system and accumulation of ROS, cells grown under dark conditions at 25°C were exposed to higher temperature of 42°C for 30 and 60 minutes (lethal temperature) as well as pretreated with 37°C for 1, 3, 6 and 24 hours were analyzed for MDA content and antioxidative enzymes. The results showed that the growth in lethal temperature was extremely poor which improved when pre-treatment of 37°C was applied before 42°C. The content of MDA in cells treated with lethal temperature was highest, which reduced when cells were pre-treated with 37°C prior to lethal stress and pre-treated cells were significantly increased DPPH radical scavenging activity, along with carotenoid contents increased in cells were pre-treatment for 24 h. The amounts of β-carotene and lutein also increased but the level of neoxanthin and violaxanthin still the same from cells were no pre-treatment, suggesting that carotenoid synthesis is stimulated upon pretreatment with mild heat stress. The expression of enzymatic antioxidant genes including, Fe superoxide dismutase (Fe SOD), Mn superoxide dismutase (Mn SOD), Glutathione peroxidase homologous (GPXH) and Glutathione S-transferase S1 (GSTS1) were up-regulated under dark condition in cells were pre-treatment and Ascorbate peroxidase (APx) were up-regulated in cells were pre-treatment for 24 h. While, Glutathione peroxidase (GPx) and Glutathione S-transferase (GST) showed increasing trend at cells were pre-treatment for 1, 3 and 6 h. These observations were attributed to heat acclimation, which improved the antioxidant defense. In vivo imaging with the Singlet Oxygen Sensor Green (SOSG) fluorescence showed mild heat and heat stress induced singlet oxygen (1O2) production in the dark condition. And hydrogen peroxide (H2O2) was observed with the highest accumulation at cell were pre-treat for 3 and 24 h. The results suggesting that singlet oxygen and hydrogen peroxide can serve a broader signaling function.