AbstractHigh temperature is a major determinant of grain growth and yield formation in wheat. The present study was undertaken to investigate the effects of high temperature regimes on the activities of key regulatory enzymes involved in starch and protein accumulation in grains of two winter wheat (Triticum aestivum L.) cultivars Yangmai 9 and Xuzhou 26 with different protein contents. Four day/night temperature regimes of 34 °C/22 °C, 32 °C/24 °C, 26 °C/14 °C and 24 °C/16 °C were established after anthesis, resulting in two daily temperature levels of 28 °C and 20 °C and two diurnal temperature differences of 12 °C and 8 °C. The activities of glutamine synthase (GS) in flag leaves and glutamate pyruvic aminotransferase (GPT), sucrose synthase (SS), soluble starch synthase (SSS) and granule‐bound starch synthase (GBSS) in grains were measured during the periods of grain filling. High temperature reduced both content and yield of starch in grains, while enhanced protein content and reduced protein yield in grains. High temperature significantly enhanced the activities of SS and GBSS on 14 days after anthesis (DAA). High temperature affected SSS slightly in Yangmai 9, but reduced SSS activity markedly in Xuzhou 26 on 14 DAA. However, at the middle and late stages of grain filling, high temperature reduced the activities of SS, GBSS and SSS significantly in the two wheat cultivars. High temperature reduced GPT activity in grains in the two wheat cultivars, but reduced GS activity in flag leaves of Yangmai 9 and enhanced GS activity of Xuzhou 26 on 14 DAA. In addition, under the same high temperature level, SS activity was higher at 34 °C/22 °C, whereas the activities of SSS and GBSS were higher at 32 °C/24 °C. Also, diurnal temperature differences affected GPT and GS activities differently between the two cultivars. Under optimum temperature level, the activities of key enzymes for starch and protein synthesis were higher at 26 °C/14 °C. The activities of SS, SSS and GBSS significantly correlated with starch accumulation in grains, except for GBSS activity to starch content on 14 DAA. GPT activity was positively correlated with protein yield, and GS activity was negatively correlated with protein yield on 14 DAA, while the activities of both GPT and GS were negatively related to protein content in grains.