In this study, Nagina22 (a heat-resistant rice variety) and YR343 (a heat-sensitive rice variety) were selected as test varieties. Six treatments were set: spray water + normal temperature treatment (NT0), spray water + high temperature stress at panicle initiation stage (HT0), and spray exogenous trehalose + high temperature stress (HT1, HT2, HT3, HT4) at 5, 10, 15, 20 mmol·L^-1, respectively. The effects of exogenous trehalose on chlorophyll content, photosynthetic gas-exchange parameters, antioxidant enzyme activities, osmotic regulatory substance content, active oxygen content and yield of rice during panicle initiation stage under high temperature stress, were analyzed to provide theoretical basis for developing heat-resistant rice cultivation and breeding heat-resistant varieties. The results show that: (1) under high temperature stress at panicle initiation stage, the contents of chlorophyll, photosynthetic gas exchange parameters and osmotic regulatory substances in the leaves of the two rice varieties decreased, while the contents of MDA and H₂O₂ in the leaves, as well as the production rate of O₂^-· increased, and the activities of antioxidant enzymes in the leaves increased first and then decreased; which resulted in a significant decline in the kernels yield and its constituent factors. (2) Application of exogenous trehalose could significantly increase the number of grains per panicles, 1000-grain weight and seed setting rate of rice at panicle initiation stage under high temperature stress, thus improving yield. The 1000-grain weight and seed setting rate of the inferior spikelets were higher than that of the superior spikelets. The optimal concentration of exogenous trehalose was 15 mmol·L^-1. (3) Exogenous trehalose spray with appropriate concentration can improve the chlorophyll content of rice leaves, promote the photosynthetic rate, enhance the activities of antioxidant enzymes SOD, POD and CAT, increase the contents of soluble sugar and soluble protein, and reduce the contents of MDA, H₂O₂ and the production rate of O₂^-·, so as to alleviate the damage to rice caused by high temperature stress at panicle initiation stage and improve the yield. It was found that exogenous application of 15 mmol·L^-1 trehalose could improve the photosynthetic physiological characteristics of rice leaves, enhance osmotic regulation ability, enhance the activity of antioxidant enzyme system, and thus improve the heat resistance and grain yield of rice under high temperature stress at the panicle initiation stage. The results can provide a theoretical basis for developing heat-resistant cultivation and breeding of heat-resistant rice varieties.