Heat stress is one of the common agrometeorological hazards in rice production in the middle and lower reaches of the Yangtze River in China. To study the mechanism of mist spray in ameliorating heat stress injury, a field experiment was conducted at Nanjing (China) with an early and a late hybrid rice varieties (Oryza sativa L.). The mist spray treatments were conducted at the flowering period, which were at August 6-10 for early rice variety and September 1-5 for late one. Four treatments at different irrigation times (T1: 08:00; T2: 12:00; T3: 14:00; CK: no mist spray; mist spray amount of 1 L·m-2) were included. The temperature and humidity at the different heights of the rice canopy and the net solar radiation above the canopy were measured. The leaf senescence, chlorophyll content, photosynthetic rate and the yields of the rice were determined. The results showed that mist spray rapidly reduced the temperature and increased the relative humidity in the canopy. The cooling effect was most significant at the top of the canopy and decreased downward from the top of canopy. The duration of the temperature decrease caused by the mist spray was 2 h. Mist spray could lead to an increase in latent heat flux (LE) and a decrease in sensible heat flux (H) in the rice field. The mist spray treatments delayed the senescence of the rice leaves, increased the activity levels of the superoxide dismutase, peroxidase, catalase, and soluble protein, reduced the malondialdehyde content, increased leaf chlorophyll content, photosynthetic rate and yield. The T2 treatment showed the most significant effect against heat stress, with the yield of the two varieties increased 13.7 and 13.6% respectively. Compared with mist spray at 08:00 or 14:00, spraying at 12:00 had the strongest resistance to heat stress in rice field.