Abstract The effects of high pressure homogenization (HPH) and/or heat treatment on the physical, structural, thermal and morphological characteristics of zein in ethanol–water solution were investigated. The results showed that HPH significantly reduced the size of zein nanoparticles. Both of HPH and heat treatment resulted in the increase of ultraviolet absorption and fluorescence intensity, as well as the enhanced thermal stability of zein. Compared with the individual HPH and thermal treatment, the coupled treatment of heat and HPH induced more obvious changes on the secondary structure of zein with the α-helix content increasing from 49.2% to 67.1%. The morphology of zein was greatly modified from sphericity to oval, dumbbell-like and random geometrical shape after HPH at 125 MPa. The synergistic effect was found between thermal treatment and HPH at 75 MPa, which resulted in size reduction of zein nanoparticles with the spherical shape and uniform distribution. Industrial relevance Thermal treatment has been extensively applied to modify the physiochemical properties of water soluble proteins like β-lactoglobulin, lactoferrin and livetins. However, the available reports on the heat-induced structural and physicochemical changes of alcohol-soluble proteins are limited. Nowadays, the new insight into protein modification is to apply physical treatments such as high pressure homogenization (HPH). HPH has been applied to modify the physiochemical, functional and structural properties of water soluble proteins, including faba bean protein, whey proteins, peanut proteins and soy protein isolate. However, little information is available about the effect of HPH treatment on structural modifications of alcohol-soluble proteins. Besides, according to the previous literatures, the combined treatment of thermal with HPH is commonly employed to improve the stability of dairy products. Nevertheless, to our best knowledge, there is no report about the coupled treatment of thermal and HPH on the modification of ethanol-soluble protein. Results from present work can be used to confirm the hypothesis that the simultaneous treatment of thermal and HPH would be an attractive modification method for zein with better thermal behaviors and structural properties to develop new food grade materials, which could be useful in the development of the potential delivery systems for bioactive compounds.