The impacts of protein types and its interaction with β-glucan on the in vitro digestibility of highland barley starch were investigated through analyzing physicochemical and microstructural properties of highland barley flour (HBF) after sequentially removing water- (WP), salt- (SP), alcohol- (AP) and alkali-soluble (AlkP) proteins. Resistant starch (RS) increased significantly in HBF after removing WP and SP, and RS of HBF was lower than that of without β-glucan. After removing WP, SP and AP, swelling powers of HBF without β-glucan (9.33–9.77) were higher than those of HBF (12.09–15.95). Trends of peak viscosity and peak temperature (thermal degradation temperature) were similar as swelling power, and HBF without AP showed the highest peak temperature (310.33 °C). Removals of different proteins improved the crystalline structure and short-range order of starch. There was a blue shift in T2 values and an opposite change in free water proportion. The matrix on starch surface was mainly formed by AP and AlkP, which could be aggregated by β-glucan. But, the inhibitory effect of AP or AlkP was stronger than that of proteins combined with β-glucan. These results help in the development of starch-based foods with different digestive properties by combining different protein types with β-glucan.