Temperature conditions affect growth and grain development during the grain-filling stage, but a comprehensive analysis of oat subjected to different temperatures during grain development has not been studied. In this study, an integrated physiological and proteomic examination of oat spikelets was performed to analyze the influence of five different day-time temperatures on stress-relative parameters and grain development. Physiological analysis showed decrease of total chlorophyll, shoot dry weight and spikelet shape development and increased activation of MDA, soluble sugar and antioxidant enzymes, with increase of temperatures. However, considering major grain yield components and storage materials, there should be an optimum temperature during ripening period. The result of proteomic analysis showed significantly high expressions of stress-related gene in high temperature treatment and grain storage materials in optimum temperature. Our findings indicate that temperature conditions during the grain-filling period exert a major influence on yield potential.