Abstract: Highland barley is a cereal crop with strong cold tolerance, short growth period, high yield and early maturity. It is the main food crop of the Tibetan people and provides an excellent source of energy and nutrition. It is rich in nutrients and physiologically active compounds, such as β-glucan, phenolic acid, flavonoids, and anthocyanins. With an increase of whole grain and flour in recent years, the market demand of highland barley products is also increasing. However, the main limitation for highland barley utilization in the food industry is its short shelf life because lipids are prone to be damaged via enzymatic hydrolysis. The release of free fatty acids (FFAs) and peroxidase promotes the oxidation of free fatty acids (FFA), causing the loss of product sensory quality and nutritional properties. For instance, several aldehydes and ketone compounds are harmful to the human body. Enzyme inactivation induced by heat treatment is a key method to prolong the shelf life of highland barley products. Various heating treatments utilize high temperature to destroy the advanced structure of the enzymes, resulting in a decrease or even inactivation of enzyme activity. The methods commonly used to inactivate lipase and lipoxygenase enzyme activity mainly include microwaving, roasting and currently emerged superheated steam treatment. The physicochemical properties of highland barley powder after different enzyme killing treatments were analyzed by differential scanning calorimetry, Fourier transform infrared spectroscopy and X-ray diffraction patterns. The results showed that the endosperm structure of highland barley was changed by three kinds of enzyme killing treatments, and the change by superheated steam treatment was the smallest. The pasteurization and thermal properties of highland barley powder were also changed by enzyme inactivation treatments. The pasteurization viscosity of highland barley powder treated by frying was the lowest and the starch structure was seriously damaged, while the viscosity of highland barley powder treated by superheated steam changed less and the degree of paste was lower than that of frying and microwave treatment. The crystallinity and enthalpy of starch molecules decreased after three kinds of enzyme killing treatment, and the short-range molecular structures of crystalline and amorphous regions were changed. The comparative analysis of three enzyme killing methods showed that superheated steam treatment induced the smallest change on the gelatinization and thermal characteristics of highland barley powder, which is conducive to the maintenance of highland barley powder quality and the development of high-quality products.