A 96-h exposure experiment was conducted to elucidate the toxicity responses of the marine diatom Thalassiosira pseudonana upon exposure to different temperatures and copper (Cu) concentrations. Three Cu treatments (seawater control; 200μg/L Cu, EC₅₀ for the yield at 25°C; and 1000μg/L Cu, EC₅₀ for growth inhibition at 25°C) were conducted against four temperatures (10°C, 15°C, 25°C and 30°C). Growth rate and photosynthetic responses showed a significant interacting thermal-chemical effect with strong synergistic responses observed at 30°C treatments. Expression of heat shock protein (hsp) was positively modulated by increasing temperatures. Hsp 90, hsp90-2 and sit1 (related to silica shell formation) were highly expressed at 30°C under 1000μg/L Cu, while the genes encoding light harvesting proteins (3HfcpA and 3HfcpB) and silaffin precursor sil3 were significantly up-regulated at 15°C under 200μg/L Cu. Our results indicated an increase Cu toxicity to T. pseudonana under high temperature and Cu dose.